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Supporting Online Material for

HSPC117 Is the Essential Subunit of a Human tRNA Splicing Ligase Complex

Johannes Popow, Markus Englert, Stefan Weitzer, Alexander Schleiffer, Beata Mierzwa, Karl Mechtler, Simon Trowitzsch, Cindy L. Will, Reinhard Lührmann, Dieter Söll,* Javier Martinez*

*To whom correspondence should be addressed. E-mail: [email protected] (J.M.); [email protected] (D.S.)

Published 11 February 2011, Science 331, 760 (2011) DOI: 10.1126/science.1197847

This PDF file includes: Materials and Methods

Figs. S1 to S10

Tables S1 to S3

References

Supporting Online Material

Materials and methods

Inter-strand ligation assay

1.11 MBq [5’-32P]cytidine-3’,5’-bisphosphate (111 TBq/mmol, Perkin Elmer) were

ligated to 50 pmol RNA oligonucleotide (5’-UCG AAG UAU UCC GCG UAC GU-

3’, obtained from IBA, Göttingen, Germany) with 20 units T4 RNA ligase 1 (NEB

Cat. No. M0204) for 1 h at 16 °C in 15 % (v/v) DMSO, 50 mM Tris-HCl pH 7.6, 10

mM MgCl2, 10 mM β-mercaptoethanol, 200 µM ATP, 0.1 mg/mL BSA in a total

reaction volume of 10 µL. Labeling reactions were stopped by addition of an equal

volume of 8 M urea, 50 mM Tris-HCl pH 8.0 and resolved by denaturing gel

electrophoresis in 10 % polyacrylamide gels (SequaGel®, National Diagnostics).

Labeled RNA was visualized by autoradiography and passively eluted from gel slices

in 300 mM NaCl at 4 °C for 12 h. RNA was precipitated by addition of 3 volumes of

ethanol and recovered by centrifugation. Labeled RNA oligonucleotides were

annealed to non-labeled complementary RNA oligonucleotides (5’-CGU ACG CGG

AAU ACU UCG A-3’, obtained from IBA, Göttingen, Germany) as follows.

Reactions containing 50 nM labeled and non-labeled complementary RNA

oligonucleotide in 30 mM HEPES-KOH pH 7.5, 2 mM MgCl2 and 100 mM KCl were

heated to 95 °C for 2 min and subsequently incubated at 37 °C for 1 h. Annealing

reactions were directly used as substrate for inter-strand ligation reactions.

2 µL of buffer LC (250 µM EDTA pH 8.0, 100 mM KCl, 3 mM MgCl2, 12.5 mM

DTT, 7.5 mM ATP, 0.5 mM GTP, 10 U/mL RNasin® (Promega Cat. No. N2611), 65

% (w/v) glycerol) were mixed with 1 µL pCp-labeled dsRNA prepared as described

above and 2 µL protein extract containing approximately 5 mg/mL protein in buffer R

(30 mM HEPES-KOH pH 7.4, 5 mM MgCl2, 100 mM KCl, 10 % (w/v) glycerol, 0.1

SOM Popow et al. 1/44

mM AEBSF, 1 mM DTT) and incubated for 30-60 min at 30 °C. Reactions were

stopped by addition of 150 µL proteinase K solution (0.3 mg/mL proteinase K,

200 mM Tris-HCl, 25 mM EDTA pH 8.0, 300 mM NaCl, 2 % SDS) and further

incubated at 65 °C for 30 min. After extraction with 150 µL

phenol/chloroform/isoamylalcohol pH 8.0 (Applichem Cat. No. A0889), RNA was

recovered from aqueous phases by precipitation with 2.5 volumes of ethanol and

analyzed by 15 % denaturing polyacrylamide gel electrophoresis. Radiolabeled RNA

was visualized by phosphorimaging.

Purification of RNA>p ligase guided by inter-strand ligation activity

All procedures described below were carried out at 4 °C. Cytoplasmic extracts were

prepared from HeLa S3 cells as described previously (S1). The cytoplasmic fraction

(approximately 5 mg/mL protein) was adjusted to 100 mM KCl, 2 mM MgCl2 and 10

% (w/v) glycerol, flash-frozen in liquid nitrogen and kept at – 80 ºC for long term

storage. 200 mL of extract were thawed and 0.176 g of powdered (NH4)2SO4 were

added per 1 mL of extract while vigorously stirring the mixture in an ice-water bath.

After the addition of (NH4)2SO4, stirring on ice was continued for 15 min. The

mixture was centrifuged (Sorvall SS34, 10400 rpm, 4 °C, 30 min), the supernatant

was recovered and filtered through a cell strainer sieve.

A Butyl Sepharose FF column (HiPrep 16/10 Butyl FF, GE Healthcare) was

equilibrated with 10 column volumes (CV) of buffer AS2000 (50 mM HEPES-KOH

pH 7.4, 2 M (NH4)2SO4, 5 mM MgCl2, 10 % (w/v) glycerol, 1 mM DTT, 0.1 mM

AEBSF) at a flow rate of 2 mL/min. The supernatant obtained after ammonium

sulphate precipitation was loaded with a flow rate of 1.5 mL/min. The column was

washed with 5 CV of AS2000 at a flow rate of 2 mL/min. A gradient to buffer AS0


(50 mM HEPES-KOH pH 7.4, 5 mM MgCl2, 10 % (w/v) glycerol, 1 mM DTT, 0.1

mM AEBSF) was applied over 10 CV and fractions of 4 mL were collected at a flow

rate of 2 mL/min. 10 µL aliquots of suitable fractions were microdialyzed against

buffer R using 0.025 µm VSWP membrane filters (Millipore Cat. No. VSWP02500)

and assayed for inter-strand ligation. Fractions exhibiting inter-strand ligation activity

contained between 1000 and 1160 mM (NH4)2SO4 and were pooled.

A desalting column (XK26/40, GE Healthcare) filled with Sephadex® G-25 Fine (GE

Healthcare Cat. No. 17-0032-01) as stationary phase was equilibrated with 5 CV

buffer BB0 (50 mM Tris-HCl pH 7.5, 3 mM MgCl2, 4 mM β-mercaptoethanol, 0.1 %

(w/v) Triton® X-100, 0.1 mM AEBSF) at 3 mL/min. The active pool obtained after

hydrophobic interaction chromatography was applied to the column and proteins were

eluted isocratically. Fractions of 3 mL were collected at a flow rate of 3 mL/min.

Proteins were detected by Bradford assay and fractions appropriately pooled.

A Blue Sepharose® column (HiTrap Blue HP 5 mL, GE Healthcare) was equilibrated

with buffer BB0 at 2 mL/min. The desalted active pool was loaded onto the column

with a flow rate of 1 mL/min. The column was washed with 10 CV of buffer BB 250

(50 mM Tris-HCl pH 7.5, 250 mM KCl, 3 mM MgCl2, 4 mM β-mercaptoethanol, 0.1

% (w/v) Triton® X-100, 0.1 mM AEBSF) and proteins were eluted with buffer

BB2000 (50 mM Tris-HCl pH 7.5, 250 mM KCl, 3 mM MgCl2, 4 mM

β-mercaptoethanol, 0.1 % (w/v) Triton® X-100, 0.1 mM AEBSF) collecting fractions

of 2 mL at a flow rate of 2 mL/min. 10 µL aliquots of suitable fractions were

microdialyzed against buffer R and assayed for inter-strand ligation. Fractions

exhibiting inter-strand ligation activity were pooled.


The desalting column described above was equilibrated with 5 CV of buffer QA (50

mM Tris-HCl pH 8.5, 3 mM MgCl2, 4 mM β-mercaptoethanol, 0.1 % (w/v) Triton®

X-100, 0.1 mM AEBSF) at 3 mL/min. The active pool obtained after dye affinity

chromatography was applied to the column and proteins were eluted isocratically.

Fractions of 2 mL were collected at a flow rate of 3 mL/min. Proteins were detected

by Bradford assay and fractions appropriately pooled.

A Mono Q column (Mono Q 5/50 GL, GE Healthcare) was equilibrated with 10 CV

of buffer QA at a flow rate of 1 mL/min. The desalted active pool obtained as

described above was loaded onto the column with a flow rate of 0.5 mL/min. Proteins

were eluted with a linear gradient to 25 % buffer QB (50 mM Tris-HCl pH 8.5, 2 M

KCl, 3 mM MgCl2, 4 mM β-mercaptoethanol, 0.1 % (w/v) Triton® X-100, 0.1 mM

AEBSF) over 10 CV collecting fractions of 0.5 mL at a flow rate of 0.5 mL/min. 10

µL aliquots of suitable fractions were microdialyzed against buffer R and assayed for

inter-strand ligation. Inter-strand ligation activity eluted between 150 and 250 mM

KCl.

Mass spectrometric analysis of proteins

For mass spectrometric analysis, protein samples were reduced with DTT, alkylated

with iodoacetamide and digested with trypsin. Tryptic peptides were resolved by

nano-HPLC and analyzed on an LTQ ion trap mass spectrometer. Mass spectrometric

data were analyzed using the MASCOT software (Matrix Science).

Cell culture, transfection and extract preparation

HeLa cells were cultured at 37 ºC, 95 % humidity and 5 % CO2 in 1X Dulbecco’s

Modified Eagle’s Medium (Invitrogen Cat. No. 10938) supplemented with 10 % fetal


bovine serum (Sigma Cat. No. F7524), 3 mM glutamine (Sigma Cat. No. G7513), 100

U/ml penicillin and 100 µg/ml streptomycin sulfate (Sigma Cat. No. P0781).

siRNAs and DNA plasmids were transfected using Lipofectamine 2000® reagent

(Invitrogen Cat. No. 11668) according to the manufacturer’s instructions. For

generation of stable cell lines, transfected cells were selected with 800 µg/mL

geneticin sulphate (Invitrogen Cat. No. 11811). When selection appeared to be

complete, single cells were seeded into 96-well plates containing growth medium

supplemented with 800 µg/mL geneticin sulphate. Single cell clones were expanded

and tested for expression of tagged proteins by Western blot.

For transfections of BAC DNA, Effectene® transfection reagent (Qiagen Cat. No.

301425) was used according to the manufacturer’s instructions. HeLa cell pools stably

transfected with dsRED-Kan/Neo-modified BAC DNA were generated by selection of

transiently transfected cells with 750 µg/mL geneticin sulphate.

Protein extracts for RNA processing assays were prepared by scraping cells on ice in

ice cold buffer R supplemented with 0.1 % (w/v) NP-40. Extracts were cleared by

centrifugation (20000 X g, 10 min, 4 °C), flash frozen in liquid nitrogen and kept at –

80 °C for long term storage.

tRNA maturation assay

A PCR product encoding pre-tRNA Phe fused to a T7 promoter was generated by

PCR amplification of Saccharomyces cerevisiae genomic DNA using the primers

ScPhe_P1 (5’-AAT TTA ATA CGA CTC ACT ATA GGG GAT TTA GCT CAG

TTG GG-3’) and ScPhe_P2 (5’-TGG TGG GAA TTC TGT GGA TCG AAC-3’). The

sequence of the amplicon was shown to correspond to S. cerevisiae tRNA3-PheGAA,

Chr. 13 (S2). Radioactively labeled pre-tRNA ScPhe was transcribed from the PCR


product using the T7 MEGAshortscript® kit (Ambion Cat. No. AM1354) adding 1.11

MBq [α-32P]guanosine-5’-triphosphate (111 TBq/mmol, Perkin Elmer) per reaction.

Transcription reactions were stopped by addition of an equal volume of 8 M urea, 50

mM Tris-HCl pH 8.0 and resolved by denaturing gel electrophoresis in 10 %

polyacrylamide gels. Labeled transcripts were visualized by autoradiography and

passively eluted from gel slices in 300 mM NaCl at 4 °C for 12 h. RNA was

precipitated by addition of 3 volumes of ethanol and recovered by centrifugation.

Labeled pre-tRNA transcripts were dissolved at 1 µM in 30 mM HEPES-KOH pH

7.5, 2 mM MgCl2 and 100 mM KCl.

Reaction mixtures containing 200 nM labeled pre-tRNA, 5 mM HEPES-KOH pH 7.4,

100 mM KCl, 5 mM MgCl2, 2 mM DTT, 5 mM ATP, 5 mM GTP and 5 mM

spermidine-HCl were heated to 95 °C for 2 min and then incubated at 25 °C for 20

min. After reannealing, RNasin was added to a final concentration of 6 U/mL. Assays

were started by mixing equal amounts of the described reaction mixture and protein

extract and incubated at 30 °C. Typically, 5 µL aliquots were withdrawn after

appropriate incubation times ranging between 10 and 60 min, added to 150 µL

proteinase K solution and incubated at 65 °C for 30 min. After extraction with 150 µL

phenol/chloroform/isoamylalcohol pH 8.0, RNA was recovered from aqueous phases

by precipitation with 2.5 volumes of ethanol and analyzed by 10 % denaturing

polyacrylamide gel electrophoresis. Radiolabeled RNA was visualized by

phosphorimaging.

tRNA exon half ligation assay

Homotetrameric tRNA splicing endonuclease from Methanocaldococcus jannaschii

was prepared essentially as described previously (S3). The hexahistidine tagged


protein was expressed from pET28a-MjSEN (gift from Tim Clausen, IMP, Vienna,

Austria) in E. coli BL21(DE3). Bacteria were grown with agitation in LB medium at

37 °C and protein expression was induced by addition of 1 mM isopropyl β-D-

thiogalactoside at O.D. (600 nm) 0.6. Cells were grown for an additional 3 h at 37 °C

and harvested by centrifugation. Bacterial pellets were resuspended in buffer MA (50

mM Tris-HCl pH 8.0, 500 mM NaCl, 10 mM imidazole-HCl pH 8.0, 0.1 % (w/v)

Tween® 20, 5 mM β-mercaptoethanol, 1 mM PMSF, 10 mL per g cell pellet weight)

and lysed by addition of 0.1 mg/mL lysozyme at 4 °C for 30 min. After sonication,

the extract was heated to 65 °C for 30 min and immediately cooled in an ice water

bath. The lysate was centrifuged (20000 X g, 4 °C, 30 min) and the supernatant

subjected to NiNTA affinity chromatography (HiTrap Chelating, GE Healthcare).

After loading, the column was washed with 10 CV of buffer MA and eluted with

buffer MB (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 250 mM imidazole-HCl pH 8.0,

0.1 % (w/v) Tween® 20, 5 mM β-mercaptoethanol). Fractions containing

endonuclease (detected by SDS-PAGE) were supplemented with 10 % (w/v) glycerol,

flash-frozen in liquid nitrogen and kept at – 80 °C for long term storage.

tRNA exon halves were prepared as described previously (S4). Radioactively labeled

hybrid pre-tRNA was transcribed from PCR templates using the T7

MEGAshortscript® kit (Ambion Cat. No. AM1354) adding 1.11 MBq [α-

32P]guanosine-5’-triphosphate (111 TBq/mmol, Perkin Elmer) per reaction. The

template PCR product was obtained by amplification of the template DNA

oligonucleotide (5’-TAA TAC GAC TCA CTA TAG CGA CCT TAG CTC AGT

TGG TAG AGC GGA GGA CTC CAG TTG CGC TTC GGC GCA GGA GAA ATC

CTT AGG TCA CTG GTT CGA-3’) with the primers Archeuka_P1 (5’-GAA ATT

AAT ACG ACT CAC TAT A-3’) and Archeuka_P2 (5’-TGG TGC GAC CTA CCG


GAT TCG AAC CAG TGA CCT A-3’). Transcription reactions were stopped by

addition of an equal volume of 8 M urea, 50 mM Tris-HCl pH 8.0 and resolved by

denaturing gel electrophoresis in 10 % polyacrylamide gels. Labeled transcripts were

visualized by autoradiography and passively eluted from gel slices in 300 mM NaCl at

4 °C for 12 h. RNA was precipitated by addition of 3 volumes of ethanol and

recovered by centrifugation.

10 pmol of hybrid pre-tRNA were cleaved in reactions (20 µL total volume)

containing 10 mM Tris-HCl pH 7.5, 100 mM KCl, 10 mM MgCl2, 1 mM DTT, 40

µM spermine and 200 ng recombinant endonuclease at 65 °C for 10 min. Cleavage

reactions were extracted with an equal volume of phenol/chloroform/isoamylalcohol

pH 8.0 and RNA was recovered from aqueous phases by precipitation with 3 volumes

of ethanol. Cleavage products were dissolved in 10 mM Tris-HCl pH 7.5, 100 mM

KOAc, 6 mM Mg(OAc)2 and 150 µM spermine-HCl pH 7.5 at a final concentration of

0.5 µM.

Ligation assays were assembled by mixing 5 µL of protein extract with 5 µL pre-

tRNA cleavage products (obtained as described above) and 10 µL buffer SB (20 mM

Tris-HCl pH 7.5, 200 mM KOAc, 12 mM Mg(OAc)2, 300 µM spermine-HCl pH 7.5,

1 mM DTT, 2 mM ATP, 1 mM GTP, 1 % (w/v) Triton® X-100, 30 % (w/v)

glycerol). Reactions were incubated for 30-60 min at 37 °C and stopped by addition

of 150 µL of proteinase K solution. After incubation at 65 °C for 30 min and

extraction with 150 µL phenol/chloroform/isoamylalcohol pH 8.0, RNA was

recovered from aqueous phases by precipitation with 2.5 volumes of ethanol and

analyzed by 10 % denaturing polyacrylamide gel electrophoresis. Radiolabeled RNA

was visualized by phosphorimaging.


Cloning of HSPC117

The coding sequence of HSPC117 (corresponding to accession number AK145916.1)

was amplified from mouse brain cDNA by polymerase chain reaction (PCR) using

Phusion® polymerase (Finnzymes Cat. No. F-530) and the primers mHSPC117_F

(5’- AAA AAG CAG GCT CCA TGA GTC GCA GCT ATA ATG A-3’) and

mHSPC117_R (5’-AGA AAG CTG GGT CCT ACT ACC CTT TAA TAA CAG

CAA TTG G-3’). The PCR product was reamplified with primers introducing

appropriate recombination cassettes and cloned into pDONR201 (Invitrogen Cat. No.

11798-014) using the Gateway BP clonase enzyme II (Invitrogen Cat. No. 11789).

HSPC117 was further recombined into the plasmid gcDNA3.1, a eukaryotic

expression vector encoding a c-myc-tag at the 5’ end of cloned genes (gift from

Hartmut Beug, IMP, Vienna, Austria) using Gateway LR clonase enzyme II

(Invitrogen Cat. No. 11791). To generate the point mutant HSPC117 C122A, an

overlap extension PCR protocol was used. Mutated HSPC117 was also recombined

into the plasmid gcDNA3.1.

Generation of sequence alignments of RtcB/HSPC117 proteins

The family was collected with an NCBI-BLASTp search (S5) within the NCBI non-

redundant protein database applying default parameters and highly significant

expectation values (< 10–30). The proteins were aligned using MAFFT (S6) and

graphically processed with ClustalX (S7).

Affinity purification of c-myc-HSPC117 from stably transfected clonal cell lines

HeLa cells stably expressing c-myc-HSPC117 or c-myc-HSPC117 C122A were

washed with PBS and harvested on ice by scraping with ice cold buffer P500 (30 mM

Tris-HCl pH 7.5, 500 mM NaCl, 2 mM MgCl2, 5 % (w/v) glycerol, 0.1 % (w/v)


Triton® X-100, 0.1 mM PMSF, 4 mM β-mercaptoethanol, supplemented with

Phosphatase Inhibitor Cocktail Set II (Merck Cat. No. 524625)). Cells were lysed by

sonication and the extract was cleared by centrifugation (20000 X g, 4 °C, 10 min).

Tagged proteins were captured with anti-c-myc agarose beads (Sigma Cat. No.

A7470) equilibrated in buffer P500 (20 µL of settled beads per confluent 15 cm cell

culture dish, 13 rpm, 4 °C, 90 min). Beads were sedimented by centrifugation and

washed three times with buffer P500 and three times with buffer P100 (30 mM Tris-

HCl pH 7.5, 100 mM NaCl, 2 mM MgCl2, 5 % (w/v) glycerol, 0.1 % (w/v) Triton®

X-100, 4 mM β-mercaptoethanol). Typically, immunoprecipitates were split in two

parts at this stage one half being used for Western blot analysis and the other half

being directly used in tRNA exon half ligation assays.

RNase T1 digest of mature tRNA

2 µM pre-tRNA (labeled with [α-32P]UTP and cleaved with MjSEN as described

above) were ligated with affinity purified c-myc-HSPC117 or with a mixture of 1

U/µL T4 RNA ligase 1 and 0.5 U/µL of T4 polynucleotide kinase (NEB Cat. No.

M0201). The ligation reaction was resolved on a 10 % denaturing gel and mature

tRNA was isolated by passive elution. RNA was precipitated with 3 volumes of

ethanol and half of the recovered RNA was digested for 90 min at 37 °C in reactions

containing 50 mM Tris-HCl pH 8.0, 2 mM EDTA and 1 U/µL RNase T1 (Roche Cat.

No. 10 109 193 001). Digested RNA was separated on a 20 % denaturing gel (17.5 cm

X 40 cm X 0.5 mm). RNase T1 fragments were visualized by autoradiography and

isolated by passive elution.


RNase T2 digest of RNase T1 fragments

An aliquot corresponding to half of the recovered RNase T1 fragments was digested

for 3 h at 37 °C in reaction mixtures containing 50 mM NaOAc pH 4.6 and 1 U/µL

RNase T2 (MoBiTec Cat. No. GE-NUC00400).

Nuclease P1 digest of circularized intron

2 µM pre-tRNA (labeled with [α-32P]ATP and cleaved with MjSEN as described

above) was ligated with affinity purified c-myc-HSPC117 or with a mixture of 1

U/µL T4 RNA ligase 1 and 0.5 U/µL of T4 polynucleotide kinase. The ligation

reaction was resolved on a 10 % denaturing gel and circularized intron was isolated

by passive elution. Circular intron was digested for 2 h at 50 °C in reactions

containing 10 mM NaOAc pH 5.2, 0.1 mM ZnCl2, 50 mM NaCl and 0.1 U/µL

nuclease P1 (Roche Cat. No. 236 225).

Thin layer chromatography

Thin layer chromatography (TLC) of RNA samples digested with RNase T2 was

carried out in solvent A (isobutyric acid/concentrated ammonia/H2O 57.7/3.8/38.5

[v/v/v]) on 10 cm X 10 cm HPTLC cellulose plates (Merck Cat. No. 1.05787).

Nuclease P1 digested RNA samples were analyzed by thin layer chromatography in

solvent B (isopropanol/concentrated HCl/H2O 70/17.5/12.5 [v/v/v]) on 20 cm X 20

cm cellulose TLC plates (Merck Cat. No. 1.05716). Radiolabeled nucleoside

monophosphate spots on TLC plates were visualized by phosphorimaging. The

identities of radiolabeled nucleoside 5’- or 3’- monophosphates were confirmed by

co-migration with non-radioactive standards (obtained as a gift from Hildburg Beier,

BEEgroup at the University of Würzburg, Würzburg, Germany) that were visualized

by UV shadowing.


Affinity purification of HSPC117 complexes with SF3b antibodies

Purification of SF3b and HSPC117-complexes was carried out essentially as

described (S8), except the anti-SF3a120 immunoaffinity chromatography step was

omitted. Briefly, spliceosomal snRNPs were depleted from HeLa nuclear extract by

immunoaffinity chromatography with the monoclonal antibody H20, which

recognizes the trimethylguanosine (m32,2,7G) snRNA cap, in buffer RC420 (20 mM

HEPES-KOH pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTE,

0.5 mM PMSF, 5% glycerol). Under these conditions the majority of SF3b dissociates

from the U2 snRNP. The flow through (nuclear extract lacking snRNPs) was adjusted

to 600 mM NaCl and applied to PAS beads covalently bound to the monoclonal

antibody 13E12 that is directed against SF3b155 (aa 99-113). The column was

washed with G600 buffer (20 mM HEPES-KOH pH 7.9, 600 mM NaCl, 1.5 mM

MgCl2, 5 % glycerol) and bound complexes were eluted with G600 buffer

supplemented with 0.1 M of peptide 155.1 (EQYDPFAEHRPPKIAC). The eluate was

concentrated by centrifugation (150000 X g, Sorvall S100-AT4, 4 °C, 5 h).

Complexes were size fractionated by gel filtration in buffer S150 (10 mM Tris-HCl

pH 7.9, 150 mM NaCl, 1 mM DTT) using a Superose 6 PC 3.2/30 column and

SMART FPLC system (GE Healthcare). Fractions were analyzed by SDS-PAGE and

proteins visualized by staining with Coomassie blue. Individual bands were excised

from the gel, digested in gel with trypsin and the extracted peptides were analyzed by

liquid chromatography-coupled tandem mass spectrometry (LC-MSMS). Proteins

were identified by searching fragment spectra of sequenced peptides against the NCBI

nr database using Mascot as search engine. Only those proteins with the highest score

for each Coomasie-stained band are indicated in Figure 3A.


Quantitative RT-PCR

RNA was prepared using Trizol® reagent (Invitrogen Cat. No. 15596) and reverse

transcribed using the First Strand cDNA Synthesis Kit (GE Healthcare Cat. No. 27-

9261-01) according to the manufacturer’s instructions. cDNA was diluted 1:100

before analysis by quantitative PCR using GoTaq® qPCR Master Mix (Promega Cat.

No. A6002). Relative expression levels were normalized using primers detecting

human cyclophylin B (CYCB). Primers were designed using the Primer3 software (v.

0.4.0); the sequences are listed below.

Transcript Primer Name Sequence

ASW (human) QPCR_HsASHWIN_F 5’-GTC CCA GGA GTT CCT TCT CC-3’

QPCR_HsASHWIN_R 5’-ACG ATG AGG GGT CTT TTC CT-3’

CGI-99 (human) QPCR_HsCGI_F 5’-GTT GAC GGC TCT CGA CTA CC-3’

QPCR_HsCGI_R 5’-TCA AAG AAC TTG GGC CAG TC-3’

CYCB (human) QPCR_HsCYCB_F 5’-GTA ATC AAG GAC TTC ATG ATC CAG GG-3’

QPCR_HsCYCB_R 5’-AAC TTT GCC AAA CAC CAC ATG CTT GC-3’

DDX1 (human) QPCR_HsDDX1_F 5’-GCA ATT GGG TCA GAT GGT CT-3’

QPCR_HsDDX1_R 5’-CTG TTC CAC CAA AGC CAA AT-3’

FAM98B (human) QPCR_HsFAM98B_F 5’-GGG AAA ACC ACT GCT GAA AA-3’

QPCR_HsFAM98B_rv 5’-TCG TTG TCT TGG GTG ACA AA-3’

HSPC117 (mouse) QPCR_MmHSPC117_F 5’-CGA CCC CAA TAA GGT CTC AC-3’

QPCR_MmHSPC117_R 5’-TTC ATG GCC TTT TCC ATA GC-3’

HSPC117 (human) QPCR_HsHSPC117_F 5’-TGA CCC CAA TAA AGT TTC TG-3’

QPCR_HsHSPC117_R 5’-ACC CTC TGG GGA AGC GAT T-3’


Construction of inducible pre-tRNA reporter constructs

A similar approach has previously been applied to study mitochondrial import of

tRNA in Trypanosoma brucei (S9). Tagged tRNA IleUAU was constructed by

introducing the mutations C28:G44 T28:A44 (as encoded e.g. in wild type tRNA7-

IleGAU, Chr. X), A32:T40 G32:C40 (as encoded e.g. in wild type tRNA153-IleAAU,

Chr. 6), U31:G41 U31:A41 (as encoded e.g. in wild type tRNA7-IleGAU, Chr. X) and

T35G (as encoded e.g. in wild type tRNA7-IleGAU, Chr. X) into tRNA10-IleUAU, Chr.

19 (S2).

For construction of the plasmid pSTet, the H1 promoter region of pSUPER Neo/GFP

(OligoEngine Cat. No. EC-PBS-0005/0006) was fused to a Tet-operator sequence

(underlined in the primer sequence) by amplification with the primers STET_P1 (5’-

CGA TAA GCT TAG ATC TCT ATC ACT GAT AGG GAA CTT ATA AGA TTC

CCA AAT CC-3’) and STET_P2 (5’-TAA AGC GCA TGC TCC-3’) as described

previously (S10). The PCR product was isolated, digested with EcoRI and BglII and

ligated into pSUPER Neo/GFP digested with the same enzymes.

To create pSTet-Ile, the annealed oligonucleotides IleTag_F (5’-GAT CCC CGC TCC

AGT GGC GCA ATC GGT TAG CGC GTG GTG CTG ATA TGA CAG TGC GAG

CGG AGC AAC ACC AAG GTT GTG AGT TCG ATC CTC ACC TGG AGC ATT

TTT A-3’) and IleTag_R (5’-AGC TTA AAA ATG CTC CAG GTG AGG ATC

GAA CTC ACA ACC TTG GTG TTG CTC CGC TCG CAC TGT CAT ATC AGC

ACC ACG CGC TAA CCG ATT GCG CCA CTG GAG CGG G-3’) corresponding

to tagged tRNA IleUAU were cloned into pSTet digested with BglII and HindIII.

The ScPhe reporter construct was designed by a similar strategy. The Saccharomyces

cerevisiae pre-tRNA Phe template sequence used for tRNA maturation assays was


fused to a Tet-inducible H1 promoter and the resulting cassette was inserted into

pDONR201 by BP recombination.

Induced expression of reporter pre-tRNAs and detection of mature tRNA by

Northern blot

HeLa T-Rex® cells (Invitrogen Cat. No. R714-07) were seeded on 6 cm culture

dishes and transfected with 200 pmol siRNAs targeting HSPC117 or EGFP. After 48

h, cells were trypsinized and seeded on new 6 cm culture dishes. 24 h later, cells were

transfected with 4 µg of reporter construct and 100 pmol of siRNAs. After 24 h, cells

from one 6 cm culture dish were trypsinized and seeded into three wells of a 6-well

plate. Immediately after seeding (48 h time point) and 24 h (24 h time point) later,

expression of tagged pre-tRNA was induced by addition of 1 µg/mL doxycycline to

wells and after 72 h RNA was isolated using Trizol® reagent. RNA isolated from un-

induced cells represented the 0 h time point.

For Northern blot analysis, 5 µg of total RNA were resolved by denaturing gel

electrophoresis in 10 % polyacrylamide gels. RNA was blotted on Hybond-N+

membranes (GE Healthcare Cat. No. RPN303B) and fixed by ultraviolet cross-

linking. Membranes were subsequently baked for 1 h at 80 °C. Blots were hybridized

with 100 pmol of a [5’-32P]labeled LNA probe detecting the 5’-exon of tagged tRNA

IleUAU (probe I5E, 5’-GG[A] TC[G] AA[C] TC[A] CA[A] CC[T] TG[G] TG[T]-3’;

LNA nucleotides are symbolized as [N]) or detecting the 3’-exon of tRNA ScPhe

(probe 5.6, 5’-TG[G] TG[G] GA[A] TT[C] TG[T] GG[A] TC[G]-3’). Equal loading

was confirmed by hybridizing the blots with a [5’-32P]labeled DNA probe detecting

U6 snRNA (5’-GCA GGG GCC ATG CTA ATC TTC TCT GTA TCG-3’).

Hybridization was carried out in 5X SSC, 20 mM Na2HPO4 pH 7.2, 7 % SDS, 1X


Denhardt’s solution and 0.1 mg/mL sonicated salmon sperm DNA (Stratagene Cat.

No. 201190) at 50 °C for DNA probes and at 80 °C for LNA probes. Blots were

washed twice for 1 min with 5X SSC, 5 % SDS and once with 1X SSC, 1 % SDS at

the respective hybridization temperatures and were visualized by phosphorimaging.

Modification of BAC RP24-139F2 for RNAi rescue experiments

The dsRED-Neo/Kan cassette was amplified from the mouse bacterial artificial

chromosome (BAC) RP23-285E19-dsRED (gift from Frank Buchholz, MPI, Dresden,

Germany) using the primers RED_F (5’-CAG GAG CTA AGG AAG CTA AAA

TGG AGA AAA AAA TCA CTG GAT ATA CCA CCG TAA CCG TAT TAC CGC

CAT GC-3’) and RED_R (5’-CCA ATA ACT GCC TTA AAA AAA TTA CGC

CCC GCC CTG CCA CTC ATC GCA GTA TCA TTT CGA ACC CCA GAG TC-

3’) (S11). The PCR product was isolated and introduced into BAC RP24-139F2

(obtained from the BACPAC Resources Center) by Red/ET recombination

(Genebridges Cat. No. K001) according to the manufacturer’s instructions. The

recombinase plasmid used was obtained as a gift from Julius Brennecke, IMBA,

Vienna, Austria. Modified BAC DNA was prepared using the Large Construct Kit

(Qiagen Cat. No. 12462) omitting the ATP-dependent exonuclease digestion step.

Inhibition of RNA>p ligase by cruciform DNA

Competing DNA duplexes were prepared by annealing the DNA oligonucleotides

PalXE_F (5’-GAT CAA CTG ATG TCG ATA TTA CGA CAT CAG TTG TAT-3’)

and PalXE_R (5’-ATA CAA CTG ATG TCG TAA TAT CGA CAT CAG TT-3’)

(S12) at 45 µM final concentration in 10 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM

MgCl2 by incubation at 90 °C for 4 min, 70 °C for 10 min and 37 °C for 15 min.

Similarly, control duplexes were obtained by annealing the DNA oligonucleotides


ScaPalΔ_F (5’-GAT CTC ATG TGT CAG TAT AAT CTG ACA CAT GA-3’) and

ScaPalΔ_R (5’-TCA TGT GTC AGA TTA TAC TGA CAC ATG A-3’). Efficient

annealing was monitored by electrophoresis in 4 % low melting agarose gels.

Inter-strand ligation reactions were set up by combining 1 μL of radiolabeled dsRNA

substrate, 2 μL of competing DNA duplex (PalXE, 45 µM) or control DNA duplex

(ScraPalΔ, 45 μM), 6 μL of buffer LC and 6 μL of HeLa cytoplasmic extract.

Incubations were performed at 30 °C and 4 µL aliquots were removed at appropriate

time points. Reactions were stopped by addition of 150 µL proteinase K solution and

further incubated at 65 °C for 30 min. After extraction with 150 µL




phosphorimaging.

tRNA maturation reactions were set up by mixing 12 μL HeLa cell extract with 12 μL

reaction mixture (100 mM KCl, 5.7 mM MgCl2, 2.5 mM DTT, 5 mM ATP, 5 mM

GTP, 6 mM spermidine‐HCl, 6 U/mL RNasin), 2 μL radiolabeled pre‐tRNA substrate

(1 µM, reannealed in 30 mM HEPES-KOH pH 7.5, 2 mM MgCl2 and 100 mM KCl),

and 4 μL annealed DNA oligonucleotides (PalXE or ScraPalΔ, 45 μM) at 30°C.

Reactions were performed at 30 °C and 5 µL aliquots were removed at appropriate

time points. Reactions were stopped by addition of 150 µL proteinase K solution and

further incubated at 65 °C for 30 min. After extraction with 150 µL





phosphorimaging.

Antibodies

A polyclonal antibody against HSPC117 was generated by immunization of rabbits

(Gramsch, Schwabhausen, Germany) with a peptide comprising amino acids 427 to

444 of human HSPC117 and affinity purification of the obtained crude serum.

Antibodies recognizing β-actin (Abcam Cat. No. ab8227), FAM98B (Sigma Cat. No.

HPA008320) and DDX1 (Bethyl Cat. No. A300-521A) were obtained from the

mentioned commercial sources. An antibody recognizing human RTCD1 was

obtained as a kind gift from Witold Filipowicz, FMI, Basel, Switzerland.

siRNAs

Silencing of human ASW (Dharmacon Cat. No. L-014299-02), human CGI-99

(Dharmacon Cat. No. L-020723-01), human DDX1 (Dharmacon Cat. No. L-011993-

01) and human FAM98B (Dharmacon Cat. No. L-018254-02) was achieved by

transfection of ON-TARGETplus® siRNA reagents. For silencing of human

HSPC117 both an ON-TARGETplus siRNA (Dharmacon Cat. No. L-017647-00) and

a set of custom designed siRNAs (siHSPC117_3UTR_P 5’-GCC CGU GCU UGU

AAA AUA AdTdT-3’ annealed to siHSPC117_3UTR_G 5’-UUA UUU UAC AAG

CAC GGG CdTdT-3’, obtained from Sigma) targeting the 3’-untranslated region of

the transcript were used.


Inpu

t

A13

A14

A15

A12

A11

B14

B15

A6 A7 A8A5A4 A10

A9A3

HSPC117

RTCD1

Ligation product

MonoQ fractionsE200 mL HeLa Extract (860 mg)

30 % AS s/n (770 mg)

Butyl Sepharose FF

Blue Sepharose HP

MonoQ 5/50 GL

AS 20000

AS 1160 - 1000 (76 mg)

BB 250 - 2000 (8 mg)

BQ 150 - 250 (260 µg)BQ 0

500

Enriched ligase fraction

BB 0 - 250

D

Figure S1.

Apyrase - - - - - - - - - + - - - - - - - - - -

DTT GTP ATP dATP Mg2+ KClCB

0 15 0 15

16 °C 30 °C

0 15 0 15 min

16 °C 30 °C

Ligation product

Substrate

Cytoplasmic Nuclear

A

OOH

OP

5’3’

5’3’

Intron

5’ Exon 3’ Exon

Pre-tRNA Exon halves

OHO

OP

5’3’

OOP

OHPP

Ligase2’-Phosphotransferase

RNA>p ligase

Endonuclease OH

O

5’3’

OP

Mature tRNA

5’3’

Mature tRNA2’

3’

P+NPP

KinaseCyclic phosphodiesterase

Yeast-/T4 Rnl1 like

5’

-Ligation product

-Substrate


0

20

40

60

80

100

120MmHSPC117

HsHSPC117

siHSPC117 (3’-UTR)siGFP

RP24-139F2 (dsRED)

Rela

tive

tran

scrip

t lev

els

[%]

B

Figure S2.

Substrate

Ligation product

siHSPC117 (3’-UTR)siGFP

RP24-139F2 (dsRED)

A

1.0 2.2 0.1 0.4 Relative intensity ligation product


249 249 249 252 250 249 220 712 224 225

CGVRLLRTNLDESDVQPVKEQLAQAMFDHIPVGVGS-KGVIPMNAKDLEEALEMGVDWS-LREGYAWAEDKEHCEEYGRMLQADPNKVSARAKKRGLPQLGTLGAGNHYAEIQVVDEIFNEYAAKKMGID CGVRLLRTNLDESDVQPVKEQLAQAMFDHIPVGVGS-KGVIPMNAKDLEEALEMGVDWS-LREGYAWAEDKEHCEEYGRMLQADPNKVSPRAKKRGLPQLGTLGAGNHYAEIQVVDEIFNEYAAKKMGID CGVRLLRTNLDESDVQPVKEQLAQSMFDHIPVGVGS-KGVIPMGAKDLEEALEMGVDWS-LREGYAWAEDKEHCEEYGRMLQADPSKVSSKAKKRGLPQLGTLGAGNHYAEVQVVDEIYDEYAAKKMGID CGVRLLRTNLTEKDVKPVKEQLAQSLFDHIPVGVGS-KGVIPMGAKELEEALEMGMDWS-LREGYAWAEDKEHCEEYGRMLQADPNKVSARAKKRGLPQLGTLGAGNHYAEIQVVDEIYNDHAAKKMGID CGVRLLRTNLYEKDVQPVKEQLAQSLFDHIPVGVGS-KGIIPMNARDLEEALEMGMDWS-LREGYVWAEDKEHCEEYGRMLNADPAKVSMRAKKRGLPQLGTLGAGNHYAEIQVVDEIYDKWSASKMGIE CGVRLLRTNLFEENVKPLKEQLTQSLFDHIPVGVGS-RGAIPMLASDLVECLEMGMDWT-LREGYSWAEDKEHCEEYGRMLQADASKVSLRAKKRGLPQLGTLGAGNHYAEVQVVDEIYDKHAASTMGID CGVRLLRTNLTEAEVGPVREQLAQALFDHIPVGVGS-QGIIPTTAKDMESALELGMDWS-LREGYAWAEDKEHCEEYGRMLNADPRYVSSRAKKRGLPQMGTLGAGNHYAEVQVVDEVYDAVAARRMGID CGVRLIRTNLTKEEVQSKIKELIKTLFKNVPSGLGS-KGILKFSKSVMDDVLEEGVRWA-VKEGYGWKEDLEFIEEHGCLKDADASYVSDKAKERGRVQLGSLGSGNHFLEVQYVEKVFDEEAAEIYGI- CGVRLIRTNLTEKEVRPRIKQLVDTLFKNVPSGVGS-QGRIKLHWTQIDDVLVDGAKWA-VDNGYGWERDLERLEEGGRMEGADPEAVSQRAKQRGAPQLGSLGSGNHFLEVQVVDKIFDPEVAKAYGL- CGVRLLASHLTLEDLLPRQKELADALYRLVPSGVGSERRDVRFSKRELKEILKEGAGWL-VKRGYGYPEDVRFIESQGRLPWANPDKVSERAFERGAPQIGTLGSGNHFLEVQYVDEVYDEEAALAFGL- CGMNALRTALTAEDLPENLAELRQAIETAVPHGRTT-GRCKRDKGAWENPPVNVDAKWAELEAGYQWLTQK-----YPRFLNTN-----------NYKHLGTLGTGNHFIEI---------------CLD 175

375 375 375 378 376 375 346 838 351 346

HKGQVCVMIHSGSRGLGHQVATDALVAMEKAMKRDKIIVNDRQLACARIASPEGQDYLKGMAAAGNYAWVNRSSMTFLTRQAFAKVFNT---TPD-DFDLHVIYDVSHNIAKVEQHVVDGKERTLLVHRK HKGQVCVMIHSGSRGLGHQVATDALVAMEKAMKRDKIIVNDRQLACARIASPEGQDYLKGMAAAGNYAWVNRSSMTFLTRQAFAKVFNT---TPD-DLDLHVIYDVSHNIAKVEQHVVDGKERTLLVHRK HKGQVCVMIHSGSRGLGHQVATDALVAMEKAMKRDKITVNDRQLACARISSDEGQDYLKGMAAAGNYAWVNRSSMTFLTRQAFSKVFNT---PPD-DLDMHVIYDVSHNIAKVEQHVVEGKEMTLLVHRK RKGQVCLMIHSGSRGLGHQVATDALVQMEKAMKRDKIEVNDRQLACARIHSQEGQDYLKAMAAAANYAWVNRSSMTFLTRQAFAKQFDT---TPD-DLDMHVIYDVSHNIAKVEEHMVDGVQKTLLVHRK EKGQVVVMIHSGSRGFGHQVATDALVQMEKAMKRDKIETNDRQLACARINSVEGQDYLKAMAAAANFAWVNRSSMTFLTRQAFAKMFNT---TPD-DLDMHVIYDVSHNIAKVENHMVDGKERKLLVHRK EEGQVVVMLHCGSRGLGHQVATDSLVEMEKAMARDGIVVNDKQLACARINSVEGKNYFSGMAAAANFAWVNRSCITFCVRNAFQKTFGM---SAD-DMDMQVIYDVSHNVAKMEEHMVDGRPRQLCVHRK TPGQVVVMIHSGSRGLGHQVATDALVAMERAMARDGIITNDRQLACARINSEEGQAYLKAMSCAANYAWVNRSSMTFLARQAFAKIFKS---TPD-DLDMHVVYDVSHNIAKVEQHCVDGQHRRLLVHRK EENQVVVLVHTGSRGLGHQICTDYLRIMEKAAKNYGIKLPDRQLACAPFESEEGQSYFKAMCCGANYAWANRQMITHWVRESFEEVFKI---HAE-DLEMNIVYDVAHNIAKKEEHIIDGRKVKVIVHRK FEGQVVVMVHTGSRGLGHQVASDYLRIMERAIRKYRIPWPDRELVSVPFQSEEGQRYFSAMKAAANFAWANRQMITHWVRESFQEVFKQ---DPEGDLGMDIVYDVAHNIGKVEEHEVDGKRVKVIVHRK FKGQVTVLIHTGSRGLGHQVCQDYVERFLKVAPRYGIELVDKQLAAAPIKSPEGQDYLQAMAAAANFAFANRQLIAHFVREAFEKV-GF---TPR-DHGLRVLYDLAHNNAKFEEH----RGRRVLVHRK ESDQVWIMLHSGSRGIGNAIGTYFIDLAQKEMQETLETLPSRDLAYFMEGTEYFDDYLKAVAWAQLFASLNRDAMMENVVTALQSITQKTVRQPQ-TLAMEEI-NCHHNYVQKEQHF----GEEIYVTRK 299

505 505 505 508 506 505 476 968 481 476

GSTRAFPPHHPLIAVDYQLTGQPVLIGGTMGTCSYVLTGTEQGMTETFGTTCHGAGRALSRAKSRRNLDFQDVLDKLADMGIAIRVASPKLVMEEAPESYKNVTDVVNTCHDAGISKKAIKLRPIAVIKG GSTRAFPPHHPLIAVDYQLTGQPVLIGGTMGTCSYVLTGTEQGMTETFGTTCHGAGRALSRAKSRRNLDFQDVLDKLADMGIAIRVASPKLVMEEAPESYKNVTDVVNTCHDAGISKKAIKLRPIAVIKG GATRAFPPHHPLIPVDYQLTGQPVLIGGTMGTCSYVLTGTDQGMTETFGTTCHGAGRALSRAKSRRNLDFQDVLDKLADLGIAIRVASPKLVMEEAPESYKNVTDVVNTCHDAGISKKAIKLRPIAVIKG GSTRAFPPHHPLIPVDYQMTGQPVLIGGTMGTCSYVLTGTESGMATTYGTTCHGAGRAWSRAKSRRNLDYQTVLKNLHELGISIRVASPKLVMEEAPESYKDVTSVVNTCHDVGISKKVLKLRPIAVIKG GSTRAFPPHHPLIPVDYQLTGQPVLVGGTMGTCSYVLTGTEQGMQETFGSTCHGAGRALSRAKSRRNLDYKDVLDKLDQLGIAIRVASPKLVMEEAPESYKDVTDVVDTCHAAGISKKCIKMRPIAVIKG GATRAFPAHHPLIPVDYQLIGQPVLIGGSMGTCSYVLTGTEQGLVETFGTTCHGAGRALSRAKSRRTITWDSVIDDLKKKEISIRIASPKLIMEEAPESYKNVTDVVDTCDAAGISKKAVKLRPIAVIKG GSTRAFPPHHPLIPADYQLIGQPVLVGGTMGTSSYVLTGTEQGFTETFGSTCHGAGRARSRNNSRNKLDYQDVLDNLKAKGIAIRVASPKLVMEEAPESYKDVSEVVDTCHQAGISKKAVKLRPIAVIKG GATRAFPPKHEAIPKEYWSVGQPVIIPGDMGTASYLMRGTEIAMKETFGSTAHGAGRKLSRAKALKLWKGKEIQRRLAEMGIVAMSDSKAVMAEEAPEAYKSVDLVADTCHKAGISLKVARMRPLGVIKG GATRAFPPGHEAVPRLYRDVGQPVLIPGSMGTASYILAGTEGAMKETFGSTCHGAGRVLSRKAATRQYRGDRIRQELLNRGIYVRAASMRVVAEEAPGAYKNVDNVVKVVSEAGIAKLVARMRPIGVAKG GATRAFGPGHPEVPEEYRRVGQPVLVPGDMGRYSYVLAGTEKAMEVSFGSSCHGAGRKMSRHQAKKVARERNLVKELAERGILVRAATRATVDEEMPEAYKDVSLVVEAVEGAGIGKKVARLRPLIVVKG GAVSA-------------RAGQYGIIPGSMGAKSFIVRGL--GNEESFCSCSHGAGRVMSRTKAKKLFSVEDQIRATAH----VECRKDAEVIDEIPMAYKDIDAVM--AAQSDLVEVIYTLRQVVCVKG 408

121 121 121 124 122 121 92 585 97 97

E.coli K12

H. sapiensM. musculusX. leavis

S. purpuratusD. melanogaster

C. elegansC. reinhardtiiM.jannaschii

P. horikoshiiT.thermophilus

[487]

WRIKKGFVPNMQVEGVFYVNDALEKLMFEELRNACRGGGVGGFLPAMKQIGNVAALPGIVHRSIGLPDVHSGYGFAIGNMAAFDMNDPEAVVSPGGVGFDI-----WRIKKGFVPNMQVEGVFYVNDALEKLMFEELRNACRGGGVGGFLPAMKQIGNVAALPGIVHRSIGLPDVHSGYGFAIGNMAAFDMNDPEAVVSPGGVGFDI-----WRIRKGFVPNMQVEGVFYVNDPLEKLMFEELRNASRGGAAGGFLPAMKQIGNVAALPGIVHRSIGLPDVHSGYGFAIGNMAAFDMENPDAVVSPGGVGFDI-----WQIKKGFVDNMKVEGRFYVDSKLEKLMFEELQQACRSKGVGGFLPAVKQIANVAALPGITGYSIGLPDIHSGYGFAIGNMAAFDMSNPEAVVSPGGVGFDI-----WRIKKGFQPNMNVEGCFYVNSRLERLMLEELKNSCRPGAVGGFLPGVKQIANVAALPGIVGRSIGLPDIHSGYGFAIGNMAAFDMNDPLSVVSPGGVGFDI-----FRIKKGFVPNMNVEGRFYVNNSLEQLMFDELKFSCDGQGIGGFLPAVRQIANVASLPGIVGHSIGLPDIHSGYGFSIGNIAAFDVGNPESVISPGGVGFDI---------------MHVPGTFYVNDALKGLLFEELQQAVVRGDHGGFLPAVKQLANVAALPGIVKRSIALPDVHSGYGFAIGNVAAFDMDNPEAVVSPGGVGFDI-----WELPKDYKDCMRVPGRIYLNEILLDELEPE---------------VLEQIANVACLPGIYKYSIAMPDVHYGYGFAIGGVAAFDQR--EGVISPGGVGFDIWEIPK-FDKRMRVPGRVYADEVLLEKMKND--------------RTLEQATNVAMLPGIYKYSIVMPDGHQGYGFPIGGVAAFDVK--EGVISPGGIGYDI-----YRIPR--QGKMRVDAVFFASKEILKDLEAE------------NYASLQQLMNVATLPGIVEPALAMPDIHWGYGFPIGGVAAFDPEE-GGVVSPGGVGFDI-----YELLT----TENAPVKMWTKGVPVEA------------------DARQQLINTAKMPFIFKHIAVMPDVHLGKGSTIGSVIPT-----KGAIIPAAVGVDI-----

N N N N N N N N N N G 77

*

E.coli K12





E.coli K12





E.coli K12





1UC2

1UC2

1UC2

1UC2

---MSRSYNDELQFLEKINKNC---MSRNYNDELQFLDKINKNC---MSRSYNDELQYLDKIHNNCMAATVREYKEELKYLNKLSDNC--MVVRPYNDELRYLEKVSDHC---MPRTFEEECDFIDRLTDTK--------------------------------MKDVLKRVSDVV----------MVVPLKRIDKIR------------MFFEKIAPYT--------------------MN

Figure S3. SOM Popow et al. 21/44

Figure S4.

A B

DDX1

c-myc-HSPC117

FAM98B

c-m

yc-W

T

c-m

yc-C

122A

IP α-c-myc

c-m

yc-W

T

c-m

yc-C

122A

c-m

yc-W

T

c-m

yc-C

122A

Buffe

r

INPUT IP α-c-myc

100 -

80 -

60 -

40 -

20 -

-Pre-tRNA

-Mature-tRNA

*

-Exon halves

-Linear intron

-Circular intron

nt


Figure S5.

MjTSEN

AOH

C C AG CC GG CAC GC G A

pU G G C C A C pU G G

pUA A

GCpU pU

G AG CG CAC C GC G AG CC GG CC G

CpUG

GA

pU GpU C

C pU C GG A G C

G

pU GA

ApUG

ApU G

A

pUG

A

A A

pUpU

pU

pU

pU

pUpU

pU

AG

c-myc-HSPC117

T4 Pnkp/Rnl1

Cp + Ap

8-mers

7-mers 2 Cp + Up

Cp + Up + Ap

6-mers

RNase T2

RNase T2

G A G G AG CC GG CC G

pU GpU C

AA

HOUpU

RNase T1

RNase T1

C pU C GG A G C

AOH



pUA A

GCpU pU

G AG CG CAC C C

CpUG

GAG

pU GA

ApUG

ApU G

AG>p

pUpU

pU

pUOH

pUpU

pU

C pU C GG A G C

AOH



pUA A

GCpU pU

G AG CG CA

CpUG

GAG

pU GA

ApUG

ApU G

pUpU

pU

pUpU

pU

pUC A

GACC

C pU C GG A G C

AOH



pUA A

GCpU pU

G AG CG CA

CpUG

GAG

pU GA

ApUG

ApU G

pUpU

pU

pUpU

pU

pUC A

GpACC

HOUCACpUG

HOAApUCCG6-mers

HOApUCCpUpUAG8-mers

7-mersHOACCpUpUAG

HOACpUCCAG

8-mers

7-mers 2 Cp + Up + Gp

6-mers Cp + Ap

Cp + Up + Ap

A

HOUCACpUG

HOAApUCCG6-mers

HOApUCCpUpUAG8-mers

7-mersHOACCpUpUAG

HOACpUCCA Gp

OH

C C G CC GG C

C GC G pA

U G G C CpA C U G G

U pA

pAG

CU U

G U UG CG C

C C GC G pAG CC GG CC G

C UG

GpA

U GU C

C U C GG pA G C

G

U G pA

pAUG

pAUG

pA

UG

pA

pApA

UU

U

U

U

UpA

pA

pA

pA

pA

G

pA

G G G G CC GG CC G

U GU C

pApA>p

HOU U

pA

pA

c-myc-HSPC117

T4 Pnkp/Rnl1 U GU G CC GG CC G

U GU C

pApA

GpA

G

pA

4 pANuclease P1

Nuclease P14 pA + pU

MjTSEN

OH

C C G CC GG C

C GC G pA

U G G C CpA C U G G

U pA

pAG

CU U

G U UG CG C

C C C

C UG

GpA

C U C GG pA G C

G

U G pA

pAUG

pAUG

pAG

UU

U

UOH

UpA

pA

pA

pA

pA

GU G CC GG CC G

U GU C

pApA

GpA

G

pUpA

B

c-m

yc-H

SPC1

17T4

Pnk

p/Rn

l1

Origin –

Gp –Up –

Cp –Ap –

6-mers 8-mers

c-m

yc-H

SPC1

17T4

Pnk

p/Rn

l1

C


Figure S6.A

α-SF3b155 13E12

Superose 6 3.2/30

G600 G600 (0.1 M peptide)

HSPC117 Complex

S150

SF3bComplex

α-m32,2,7G H20

RC420 Bound RC420 Flowthroughadjusted to 600 mM NaCl

HeLa nuclear Extract

Mar

ker

Subs

trat

e

Inpu

t

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

100 -

80 -

70 -

60 -

40 -

20 -

90 -

50 -

30 -

siHSPC117 plus Superose 6 Fractions

*

- Mature tRNA

- Pre-tRNA

- Exon halves

*

*

B

nt


A

- Ligation product

- Substrate

GST-RtcA

ATP

1

Subs

trat

e

2

––

3

–+

5

++

4

+–

HSPC117 Complex B

Figure S7.

- Mature tRNA

- Pre-tRNA

- Exon halves

Mar

ker

Subs

trate

No NTP

-Apy

rase

30

min

30

°C+d

ATP

1 2 3 4 5 876

+AM

PcPP

+AM

PPNP

+ATP

γS

HSPC117 Complex

- Linear intron

- Circular intron

9 121110

+Apy

rase

30

min

30

°C

+ATP

+AM

PPcP

-Mg

2+

*

100 -

80 -

70 -

40 -

20 -

nt

No NTP 41 % 20 %– Apyrase 11 11+ Apyrase 15 11ATP 100 100dATP 89 97AMPcPP 40 48AMPPNP 10 10ATPγS 78 92AMPPcP 0 7No Mg 5 16

Exon

hal

f lig

atio

nIn

tron

circu

lariz

atio

n

2+


siEGFPUntreated

siHSPC117siASW

siCGI99siDDX1

siFAM98B

-+

-----

+-

-----

--

+----

--

-++--

--

-+-+-

--

-+--+

--

--++-

--

--+-+

--

---++

--

-+++-

--

-++-+

--

-+-++

--

--+++

-DDX1

-HSPC117

=FAM98B

-β-Actin

Figure S8.

A

siASW siASW siASW siCGI99B

C

siEGFPUntreated siHSPC117siASW

siCGI99siASW

siDDX1siASW

siFAM98BsiCGI99siDDX1

siCGI99siFAM98B

siDDX1siFAM98B

Subs

trat

e10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 min

100 -

80 -

60 -

40 -

- Pre-tRNA

- Mature tRNA

- Exon halves

*

Mar

ker

0.3 0.6 0.8 0.4 0.7 1.0 0.1 0.2 0.2 0.3 0.6 0.7 0.4 0.7 1.0 0.3 0.5 0.8 0.3 0.5 0.6 0.4 0.5 0.7 0.4 0.7 0.8 Relative intensity mature tRNA

siEGFPUntreated siHSPC117siCGI99

Subs

trat

e

10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 minsiDDX1

siCGI99siFAM98B

siDDX1siFAM98B

siDDX1siFAM98B

100 -

80 -

60 -

40 -

- Pre-tRNA

- Mature tRNA

- Exon halves

*

Mar

ker

0.3 0.6 0.8 0.4 0.7 1.0 0.2 0.2 0.2 0.3 0.5 0.7 0.3 0.6 0.7 0.4 0.8 0.9 0.5 0.7 0.9 Relative intensity mature tRNA

nt

nt


Figure S9.

A105 20 105 20

min

PalXE ScraPalΔ

*

- Ligation Product

- Substrate

B

10 20 300 60 m

in

10 20 300 60

PalXE ScraPalΔ

*

- Pre-tRNA

- Mature tRNA

- Exon halves

0.0 0.1 0.2 0.3 0.6 0.1 0.3 0.5 0.7 1.0 Relative intensity mature tRNA


Figure S10.

Homo_sapiens_chr6.trna165-IleA GGCCGGTTAGCTCAGTTGGTCAGAGCGTGGTGCTAATA------------Homo_sapiens_chr6.trna28-IleAA GGCCGGTTAGCTCAGTTGGTCAGAGCGTGGTGCTAATA------------Homo_sapiens_chrX.trna5-IleGAT GGCCGGTTAGCTCAGTTGGTAAGAGCGTGGTGCTGATA------------Homo_sapiens_chrX.trna6-IleGAT GGCCGGTTAGCTCAGTTGGTAAGAGCGTGGTGCTGATA------------Homo_sapiens_chrX.trna7-IleGAT GGCCGGTTAGCTCAGTTGGTAAGAGCGTGGTGCTGATA------------Homo_sapiens_chrX_random.trna1 GGCCGGTTAGCTCAGTTGGTAAGAGCGTGGTGCTGATA------------Homo_sapiens_chrX_random.trna2 GGCCGGTTAGCTCAGTTGGTAAGAGCGTGGTGCTGATA------------Homo_sapiens_chrX_random.trna3 GGCCGGTTAGCTCAGTTGGTAAGAGCGTGGTGCTGATA------------Homo_sapiens_chrX_random.trna4 GGCCGGTTAGCTCAGTTGGTAAGAGCGTGGTGCTGATA------------Homo_sapiens_chrX_random.trna5 GGCCGGTTAGCTCAGTTGGTAAGAGCGTGGTGCTGATA------------Homo_sapiens_chr17.trna34-IleA GGCCGGTTAGCTCAGTTGGTTAGAGCGTGGTGCTAATA------------Homo_sapiens_chr6.trna163-IleA GGCCGGTTAGCTCAGTTGGTTAGAGCGTGGTGCTAATA------------Homo_sapiens_chr6.trna59-IleAA GGCCGGTTAGCTCAGTTGGTTAGAGCGTGGTGCTAATA------------Homo_sapiens_chr14.trna10-IleA GGCCGGTTAGCTCAGTTGGTTAGAGCGTGGTGCTAATA------------Homo_sapiens_chr17.trna9-IleAA GGCCGGTTAGCTCAGTTGGTTAGAGCGTGGTGCTAATA------------Homo_sapiens_chr6.trna11-IleAA GGCCGGTTAGCTCAGTTGGTTAGAGCGTGGTGCTAATA------------Homo_sapiens_chr6.trna154-IleA GGCCGGTTAGCTCAGTTGGTTAGAGCGTGGTGCTAATA------------Homo_sapiens_chr6.trna80-IleAA GGCCGGTTAGCTCAGTTGGTTAGAGCGTGGCGCTAATA------------Homo_sapiens_chr6.trna158-IleA GGCCGGTTAGCTCAGTTGGTTAGAGCGTGGTGCTAATA------------Homo_sapiens_chr6.trna57-IleAA GGCCGGTTAGCTCAGTCGGCTAGAGCGTGGTGCTAATA------------Homo_sapiens_chr6.trna153-IleA GGCTGGTTAGCTCAGTTGGTTAGAGCGTGGTGCTAATA------------Homo_sapiens_chr6.trna38-IleAA GGCTGGTTAGTTCAGTTGGTTAGAGCGTGGTGCTAATA------------Homo_sapiens_chr6.trna29-IleTA GCTCCAGTGGCGCAATCGGTTAGCGCGCGGTACTTATATGGCAGTATGTGHomo_sapiens_chr6.trna63-IleTA GCTCCAGTGGCGCAATCGGTTAGCGCGCGGTACTTATAAGACAGTGCACCHomo_sapiens_chr6.trna55-IleTA GCTCCAGTGGCGCAATCGGTTAGCGCGCGGTACTTATACAACAGTATATGHomo_sapiens_chr2.trna5-IleTAT GCTCCAGTGGCGCAATCGGTTAGCGCGCGGTACTTATACAGCAGTACATGHomo_sapiens_chr19.trna10-IleT GCTCCAGTGGCGCAATCGGTTAGCGCGCGGTACTTATATGACAGTGCGAGIleMut GCTCCAGTGGCGCAATCGGTTAGCGCGTGGTGCTGATATGACAGTGCGAG G T G CA T GG AG GCG*GG *CT*ATA

--------ACGCCAAGGTCGCGGGTTCGATCCCCGTACGGGCCA--------ACGCCAAGGTCGCGGGTTCGATCCCCGTACGGGCCA--------ACACCAAGGTCGCGGGCTCGACTCCCGCACCGGCCA--------ACACCAAGGTCGCGGGCTCGACTCCCGCACCGGCCA--------ACACCAAGGTCGCGGGCTCGACTCCCGCACCGGCCA--------ACACCAAGGTCGCGGGCTCGACTCCCGCACCGGCCA--------ACACCAAGGTCGCGGGCTCGACTCCCGCACCGGCCA--------ACACCAAGGTCGCGGGCTCGACTCCCGCACCGGCCA--------ACACCAAGGTCGCGGGCTCGACTCCCGCACCGGCCA--------ACACCAAGGTCGCGGGCTCGACTCCCGCACCGGCCA--------ACGCCAAGGTCGCGGGTTCGAACCCCGTACGGGCCA--------ACGCTAAGGTCGCGGGTTCGATCCCCGTACTGGCCA--------ACGCCAAGGTCGCGGGTTCGATCCCCGTACTGGCCA--------ACGCCAAGGTCGCGGGTTCGATCCCCGTACGGGCCA--------ACGCCAAGGTCGCGGGTTCGATCCCCGTACGGGCCA--------ACGCCAAGGTCGCGGGTTCGATCCCCGTACGGGCCA--------ACGCCAAGGTCGCGGGTTCGATCCCCGTACGGGCCA --------ACGCCAAGGTCGCGGGTTCGATCCCCGTACGGGCCA--------ACGCCAAGGTCGCGGGTTCGATCCCCGTACGGGCCA--------ACGCCAAGGTCGCGGGTTCGATCCCCGTACGGGCCA--------ACGCCAAGGTCGCGGGTTCGATCCCCGTACTGGCCA--------ACGCCAAGGTCGTGGGTTCGATCCCCATATCGGCCATGCGAGTGATGCCGAGGTTGTGAGTTCGAGCCTCACCTGGAGCATGTGAGCAATGCCGAGGTTGTGAGTTCAAGCCTCACCTGGAGCA TGCGGGTGATGCCGAGGTTGTGAGTTCGAGCCTCACCTGGAGCA CAGAGCA-ATGCCGAGGTTGTGAGTTCGAGCCTCACCTGGAGCACGGAGCA-ATGCCGAGGTTGTGAGTTCGATCCTCACCTGGAGCACGGAGCA-ACACCAAGGTTGTGAGTTCGATCCTCACCTGGAGCA

A**C *AGGT G G G TC A C C G CA

A

intron

Dcontrol

siRNA

0

20

40

60

80

100

Rela

tive

tran

scrip

t lev

els

[%]

B

Seed

cells

+ siR

NA: E

GFP/H

SPC1

17

Split

cells

+ siR

NA: E

GFP/H

SPC1

17

+ p

STet

Ile/

pDONR2

01 S

cPhe

RNA

isola

tion

+DOX

for 4

8 h

+DOX

for 2

4 h

24 h 48 h 24 h 48 h24 h

Split

cells

C

TTTTTH1 promoter TetO 5’-exon intron 3’-exon* * * * *

I5E

pSTet*

TTTTTH1 promoter TetO 5’-exon intron 3’-exon

5.6

pDONR201

UAU(Ile )

(ScPhe)


Supplemental figure legends

Fig. S1. RNA ligation mechanisms and properties of interstrand ligase activity. (A)

Scheme illustrating mechanistic differences in described RNA ligation pathways. (B)

An equal amount of cytoplasmic or nuclear extract was incubated with 3’-P, 5’-OH

dsRNA substrates at 16 °C or 30 °C in presence of ATP. Aliquots of reactions were

withdrawn at indicated time points and analyzed by denaturing gel electrophoresis.

(C) Cytoplasmic extracts were incubated with 3’-P, 5’-OH dsRNA substrates in

absence or presence of DTT (0.5-15 mM), GTP (0-1 mM), ATP (0-3 mM), Apyrase

(1 mU), dATP (0-3 mM), MgCl2 (1-10 mM) and KCl (50-150 mM). Aliquots of

reactions were withdrawn at indicated time points and analyzed by denaturing gel

electrophoresis. (D) Fractionation scheme established for the partial purification of

RNA>p ligase from HeLa extracts. Throughout the purification inter-strand ligation

of [5’-32P]pCp radiolabeled 5’-OH, 3’-P dsRNA was used to monitor RNA>p ligase

activity. (E) Co-fractionation of HSPC117 and RTCD1 throughout the partial

purification of interstrand ligase activity. Aliquots of fractions of the final MonoQ

column were assayed for interstrand ligase activity (bottom panel) and in parallel

analyzed by Western blot (upper panels).

Fig. S2. Mouse BAC transgenic rescue (S11) of the in vitro RNA ligation defect in

extracts depleted of HSPC117 by RNAi. (A) Stable HSPC117 dsRED-mouse-BAC

transgenic HeLa cell pools or wild type HeLa cells were transfected with siRNA

targeting the non-conserved 3’-UTR of the human HSPC117 mRNA or control

siRNAs. Extracts prepared from these cells were assayed for inter-strand ligation.

Numbers below lanes indicate relative amounts of ligation product. The signal in lane

1 was arbitrarily set to 1.0. (B) RNA was isolated from the same experiments in

parallel and analyzed for levels of mouse and human HSPC117 mRNA by qRT-PCR.


Expression levels of human HSPC117 in HeLa cells and human and murine HSPC117

in mouse-BAC transgenic HeLa cell pools were arbitrarily set to 100 %.

Fig. S3. Sequence alignment of HSPC117/RtcB proteins. HSPC117/RtcB proteins are

widely spread in archaea, bacteria and animals, but not in plants and fungi. The

following sequences (including NCBI protein accessions) were used: Homo sapiens

(AAH00151.1), Mus musculus (NP_663397.1), Xenopus laevis (NP_001090369.1),

Strongylocentrotus purpuratus (XP_793085.2), Drosophila melanogaster

(NP_609965.1), Caenorhabditis elegans (NP_492498.1), Chlamydomonas reinhardtii

(XP_001699521.1), Methanocaldococcus jannaschii DSM 2661 (NP_247666.1),

Pyrococcus horikoshii (pdb|1UC2|A), Thermus thermophilus HB8 (YP_145051.1),

Escherichia coli str. K12 (NP_417879.1). In the M. jannaschii protein sequence, the

487 residues of the unaligned intein domain were deleted. Below the aligned

sequences, the alpha helices and beta strands inferred from the P. horikoshii RtcB

crystal structure are represented in red and green, respectively (S13). The asterisk

indicates the position of the characterized C122A mutation in the presumed active site

of human/murine HSPC117. Recently, the corresponding cysteine residue has been

demonstrated to be essential for the metal ion dependent RNA>p ligase activity of

archaeal RtcB proteins (S14).

Fig. S4. Confirmation of interactions between WT and C122A mutant HSPC117,

DDX1 and FAM98B. (A) Immunoprecipitates of WT or C122A c-myc-HSPC117

were analyzed by Western blot for the presence of DDX1 and FAM98B. (B) In

parallel, these immunoprecipitates were incubated with tRNA exon halves to assess

RNA ligase activity.


Fig. S5. Detailed explanation of the protocol used for nearest neighbor analysis of the

splice junction phosphate. (A) The depicted pre-tRNA is body-labeled with [α-

32P]UTP. For simplicity only radiolabeled phosphates are shown. Cleavage of this

pre-tRNA with recombinant splicing endonuclease of Methanocaldococcus jannaschii

(MjTSEN) yields 5’-exon halves with a radiolabeled terminal 2’,3’-cyclic phosphate.

Ligation of the exon halves with affinity purified c-myc-HSPC117 leads to formation

of mature tRNA molecules containing the precursor-derived splice junction

phosphate. During ligation of the same exon halves with a mixture of T4 Pnkp and T4

Rnl1 the splice junction phosphate is replaced by non-radioactive exogenous

phosphate provided by ATP present in the reaction mixture. As a consequence, the

radioactive splice junction label is lost. Mature tRNA is isolated from both ligation

reactions and digested with RNase T1. The splice junction is now contained in an

RNase T1 7-mer which can be isolated by preparative denaturing gel electrophoresis.

Complete digestion of the RNase T1 7-mers with RNase T2 releases the splice

junction phosphate as a guanosine 3’-monophosphate (Gp). Therefore, detection of

radiolabeled Gp indicates incorporation of the precursor-derived, radiolabeled

terminal 2’,3’-cyclic phosphate into the splice junction as a 3’,5’-phosphodiester. (B)

The depicted pre-tRNA is body-labeled with [α-32P]ATP. Cleavage of this precursor

produces linear intron with a radiolabeled terminal 2’,3’-cyclic phosphate. Ligation of

this linear intron with affinity purified c-myc-HSPC117 yields a circularized intron

containing the radiolabeled terminal phosphate. During ligation of linear intron with

T4 Pnkp and T4 Rnl1 this phosphate is removed and an exogenous phosphate

provided by non-radioactive ATP present in the reaction mixture is deposited at the

5’-OH of the first uridine nucleotide of the linear intron. Circularized intron is isolated

by preparative denaturing gel electrophoresis. During ligation with affinity purified c-


myc-HSPC117 the radiolabeled 2’,3’-cyclic phosphate becomes the 5’-phosphate of

the first uridine nucleotide of the linear intron. Digestion of circularized intron with

nuclease P1 therefore releases the junction phosphate as uridine 5’-monophosphate

(pU). Consequently, detection of radiolabeled pU indicates incorporation of the

radiolabeled terminal 2’,3’-cyclic phosphate of the linear intron into circular intron as

a 3’,5’-phosphodiester. (C) RNase T1 fragments derived from [α-32P]UTP-

radiolabeled mature tRNA generated either by T4 Pnkp/Rnl1 or affinity purified c-

myc-HSPC117 were resolved by denaturing gel electrophoresis. The 6 and 8-mers

were isolated from the gel, digested by RNase T2 and analyzed by TLC in solvent A

to confirm the identity of the analyzed RNA products.

Fig. S6. Purification and properties of HSPC117 complexes co-selected with

monoclonal SF3b antibodies. (A) Protocol for the purification of SF3b complexes

from HeLa nuclear extracts. Spliceosomal snRNPs were removed from the latter by

immobilized monoclonal antibody H20 recognizing the trimethylguanosine (m32,2,7G)

snRNA cap; HSPC117 and SF3b complexes were affinity purified via immobilized

anti-SF3b155 monoclonal antibody 13E12. (B) HSPC117-complexes co-selected

together with SF3b complexes biochemically rescue the in vitro tRNA ligation defect

in extracts depleted of HSPC117 by RNAi. Extracts of cells depleted of HSPC117 by

RNAi were complemented by Superose 6 fractions containing the HSPC117-complex

co-selected together with SF3b particles. Fractions were assayed for processing of

pre-tRNA. Asterisks denote unrelated bands.

Fig. S7. Characterization of properties and cofactor requirements of HSPC117

complexes co-selected with monoclonal SF3b antibodies. (A) Inter-strand ligation by

the HSPC117 complex is dependent on RNA 3’-P terminal cyclase. The HSPC117-


complex co-selected with SF3b particles was incubated with 3’-P, 5’-OH dsRNA

substrates in presence of ATP (lanes 3 and 5) or recombinantly expressed, GST-

tagged RtcA from E. coli (lanes 4 and 5). (B) ATP and β,γ-hydrolyzable ATP

analogues stimulate RNA ligase activity of the HSPC117 complex. The HSPC117-

complex co-selected with SF3b particles was incubated with tRNA halves without any

addition of nucleoside triphosphate cofactors (lane 3), after pre-incubation in presence

or absence of apyrase (1 mU, lanes 4 and 5), in presence of ATP (1 mM, lane 6),

dATP (1 mM, lane 7), adenosine-5’-[(α,β)-methyleno]triphosphate (AMPcPP, 1 mM,

lane 8), adenosine-5’-[(β,γ)-imido]triphosphate (AMPPNP, 1 mM, lane 9), adenosine-

5’-(γ-thio)-triphosphate (ATPγS, 1 mM, lane 10), adenosine-5’-[(β,γ)-

methyleno]triphosphate (AMPPcP, 1 mM, lane 11) or without addition of magnesium

ions (lane 12). Activity signals were quantified using ImageQuant and are displayed

as relative values. The activity signals obtained in presence of ATP were arbitrarily

set to 100 %.

Fig. S8. Effects of simultanous depletion of HSPC117-interacting proteins on tRNA

maturation in cell extracts. (A) Protein extracts prepared from HeLa cells depleted of

binary combinations of the identified HSPC117 interacting proteins were assayed for

maturation of pre-tRNA transcripts. (B) Protein extracts prepared from HeLa cells

depleted of ternary combinations of the identified HSPC117 interacting proteins were

assayed for maturation of pre-tRNA transcripts. EGFP and HSPC117 were targeted as

negative or positive controls, respectively. Asterisks denote unrelated bands. Numbers

below lanes indicate relative amounts of mature tRNA. The signal obtained in the

control transfection (siEGFP) after 30 min was arbitrarily set to 1.0. (C) The extracts

were analyzed by Western blot for the presence of HSPC117, DDX1 and FAM98B.


Fig. S9. Interfering with RNA>p ligase by specific inhibition with cruciform DNA. In

accord with previous studies describing the interaction of HSPC117 complexes with

cruciform DNA structures (S12), inter-strand ligation and tRNA maturation are

specifically inhibited by addition of cruciform but not control dsDNA. (A) HeLa cell

extracts were assayed for inter-strand ligation in presence of a 2000-fold molar excess

of cruciform (ScraPal) or control DNA (ScraPalΔ) duplexes over dsRNA substrate.

Aliquots of reactions were withdrawn at indicated time points and analyzed by

denaturing gel electrophoresis. (B) HeLa extracts were assayed for tRNA maturation

in presence of a 100-fold molar excess of cruciform (ScraPal) or control (ScraPalΔ)

DNA duplexes over RNA substrate. Aliquots of reactions were withdrawn at

indicated time points and analyzed by denaturing gel electrophoresis. Numbers below

lanes indicate relative intensities of mature tRNA. The signal obtained after 60 min

incubation in presence of unspecific competitor DNA was arbitrarily set to 1.0. The

asterisk denotes an unrelated band.

Fig. S10. Design of tagged tRNA Ile and validation of RNAi-mediated depletion of

HSPC117 for in vivo tRNA processing experiments. (A) Alignment of the pSTet

insert encoding tagged pre-tRNA IleUAU with annotated human genomic tRNA Ile loci

(S2). Capital letters in the bottom line indicate universally conserved residues,

asterisks indicate mutated positions with respect to tRNA10-IleUAU, Chr. 19. All

introduced mutations comprise nucleotides and base pairs present in human genomic

sequences of isoacceptor tRNAs. Underlined bold letters indicate the hybridization

region of probe I5E. (B) Schematic representation of elements present in the tRNA

reporter constructs. Asterisks symbolize introduced mutations for pre-tRNA IleUAU;

the bar indicates the hybridization region of probes I5E and 5.6. (C) Experimental

timeline for transfection of siRNAs and reporter constructs and induction of tagged


tRNA transcription. (D) Validation of RNAi-mediated depletion of HSPC117 in HeLa

T-Rex® cells by quantitative PCR (N = 3). Values are depicted as mean +/- SD.


Supplemental tables

Table S1. Proteins identified by in solution tryptic digest and MS analysis of MonoQ

fraction enriched for RNA>p ligase.

Gene Symbol

MW [kDa]

Number of Unique

Peptides

Entrez Gene-ID Gene Description

EEF2 95 34 1938 eukaryotic translation elongation factor 2 MSN 68 34 4478 moesin

ELAC2 92 28 60528 elaC homolog 2 (E. coli) CCT2 57 23 10576 chaperonin containing TCP1, subunit 2 (beta) CCT8 59 22 10694 chaperonin containing TCP1, subunit 8 (theta)

HSPA8 71 22 3312 heat shock 70kDa protein 8 PKM2 60 20 5315 pyruvate kinase, muscle

SYNCRIP 63 20 10492 synaptotagmin binding, cytoplasmic RNA interacting protein

NARS 63 19 4677 asparaginyl-tRNA synthetase PUS7L 82 17 83448 pseudouridylate synthase 7 homolog (S. cerevisiae)-

like SND1 100 16 27044 staphylococcal nuclease and tudor domain containing

1 CCT5 59 15 22948 chaperonin containing TCP1, subunit 5 (epsilon)

HSPA1B 70 15 3304 heat shock 70kDa protein 1B RDX 69 15 5962 radixin

CACYBP 26 14 27101 calcyclin binding protein CCT6A 58 14 908 chaperonin containing TCP1, subunit 6A (zeta 1) DUS3L 72 14 56931 dihydrouridine synthase 3-like (S. cerevisiae) G6PD 59 14 2539 glucose-6-phosphate dehydrogenase

GLT25D1 74 14 79709 glycosyltransferase 25 domain containing 1 NMT1 57 14 4836 N-myristoyltransferase 1 CCT3 60 13 7203 chaperonin containing TCP1, subunit 3 (gamma)

FARSB 66 13 10056 phenylalanyl-tRNA synthetase, beta subunit MTHFD1 102 13 4522 methylenetetrahydrofolate dehydrogenase (NADP+

dependent) 1 ACLY 121 12 47 ATP citrate lyase EIF4A3 47 12 9775 eukaryotic translation initiation factor 4A, isoform 3 GARS 83 12 2617 glycyl-tRNA synthetase LGTN 65 12 1939 ligatin SSB 47 12 6741 Sjogren syndrome antigen B

TCP1 60 12 6950 t-complex 1 ABCE1 67 11 6059 ATP-binding cassette, sub-family E, member 1 HSPA9 74 11 3313 heat shock 70kDa protein 9 IDH1 47 11 3417 isocitrate dehydrogenase 1

BASP1 23 10 10409 brain abundant, membrane attached signal protein 1 FAM129B 83 10 64855 family with sequence similarity 129, member B

FKBP4 52 10 2288 FK506 binding protein 4 GMPS 77 10 8833 guanine monphosphate synthetase

BLVRA 33 9 644 biliverdin reductase A


Table S1. (continued)

Gene Symbol

MW [kDa]

Number of Unique

Peptides


C22orf28 55 9 51493 chromosome 22 open reading frame 28 LRRC40 68 9 55631 leucine rich repeat containing 40 METTL1 34 9 4234 methyltransferase like 1 PAPSS2 71 9 9060 3'-phosphoadenosine 5'-phosphosulfate synthase 2 TSFM 42 9 10102 Ts translation elongation factor, mitochondrial CCT7 55 8 10574 chaperonin containing TCP1, subunit 7 (eta)

SERBP1 45 8 26135 SERPINE1 mRNA binding protein 1 SLC9A3R1 39 8 9368 solute carrier family 9 (sodium/hydrogen exchanger),

member 3 regulator 1 PRPS1 35 7 5631 phosphoribosyl pyrophosphate synthetase 1 SHMT2 55 7 6472 serine hydroxymethyltransferase 2 (mitochondrial)

CWF19L1 61 6 55280 CWF19-like 1 DDX1 75 6 1653 DEAD (Asp-Glu-Ala-Asp) box polypeptide 1

NAMPT 56 6 10135 nicotinamide phosphoribosyltransferase PRPSAP2 47 6 5636 phosphoribosyl pyrophosphate synthetase-associated

protein 2 TWF1 48 6 5756 twinfilin homolog 1 (Drosophila)

ANKZF1 81 5 55139 ankyrin repeat and zinc finger domain containing 1 CALD1 94 5 800 caldesmon 1 EIF4B 69 5 1975 eukaryotic translation initiation factor 4B EIF4H 27 5 7458 eukaryotic translation initiation factor 4H FARSA 57 5 2193 phenylalanyl-tRNA synthetase, alpha subunit GART 108 5 2618 phosphoribosylglycinamide formyltransferase

PAPSS1 71 5 9061 3'-phosphoadenosine 5'-phosphosulfate synthase 1 RNASEH2B 35 5 79621 ribonuclease H2, subunit B

RPRD1B 37 5 58490 regulation of nuclear pre-mRNA domain containing 1B

RRM1 90 5 6240 ribonucleotide reductase M1 WDR4 45 5 10785 WD repeat domain 4 ATE1 55 4 11101 arginyltransferase 1

CMPK1 15 4 51727 cytidine monophosphate (UMP-CMP) kinase 1, cytosolic

CPS1 165 4 1373 carbamoyl-phosphate synthetase 1, mitochondrial EEF1A1 50 4 1915 eukaryotic translation elongation factor 1 alpha 1 ERP29 27 4 10961 endoplasmic reticulum protein 29

MAT2A 44 4 4144 methionine adenosyltransferase II, alpha NME1-NME2

17 4 654364 NME1-NME2 readthrough

PPWD1 74 4 23398 peptidylprolyl isomerase domain and WD repeat containing 1

RNASEH2C 18 4 84153 ribonuclease H2, subunit C UBE2N 17 4 7334 ubiquitin-conjugating enzyme E2N

CHMP2A 25 3 27243 chromatin modifying protein 2A DUS2L 55 3 54920 dihydrouridine synthase 2-like

FAM98B 46 3 283742 family with sequence similarity 98, member B



Gene Symbol

MW [kDa]

Number of Unique

Peptides


HNRNPK 51 3 3190 heterogeneous nuclear ribonucleoprotein K HSPA14 55 3 51182 heat shock 70kDa protein 14 LRPAP1 41 3 4043 low density lipoprotein receptor-related

proteinassociated protein 1 PFKM 85 3 5213 phosphofructokinase, muscle PUS7 27 3 54517 pseudouridylate synthase 7 homolog (S. cerevisiae)

RNASEH2A 33 3 10535 ribonuclease H2, subunit A TRMT112 14 3 51504 tRNA methyltransferase 11-2 homolog (S. cerevisiae) WBSCR22 32 3 114049 Williams Beuren syndrome chromosome region 22 AKR7A2 40 2 8574 aldo-keto reductase family 7, member A2

C14orf166 27 2 51637 chromosome 14 open reading frame 166 PAWR 37 2 5074 PRKC, apoptosis, WT1, regulator RAN 26 2 5901 RAN, member RAS oncogene family

RANBP1 32 2 5902 RAN binding protein 1 RDBP 43 2 7936 RD RNA binding protein

UBE2V1 19 2 7335 ubiquitin-conjugating enzyme E2 variant 1


Comments

Interacts with cell adhesion proteins

(S15, S16), essential for embryonal

development in mice (S17)

Interacts with ninein (S18)

Function in RNA

metabolism

(S19)

(S20)

Expression in human

tissues

++

++

+

++

+

UniGene Accession

Hs.474643

Hs.440599

Hs.6799

Hs.534457

Hs.549577

Domain description

Uncharacterized protein family

UPF0027

DEAD/DEAH box helicase

SPRY domain, unknown function

Helicase conserved C-

terminal domain

Domain of unknown function

Putative carnitine deficiency-

associated protein, function unknown

From to (a.a.)

65-505

26-70, 538-609

130-243

538- 609

16-330

1- 244

Pfam-domains (S21)

UPF0027

DEAD

SPRY

Helicase_C

DUF2465

RLL

Representative protein

(Accession)

NP_055121

NP_004930

NP_775882

NP_057123

NP_076998

Aliases

DJ149A16.6; RP1-149A16.6

DBP-RB; UKVH5d

FLJ38426

CLE; CLE7; RLLM1; LCRP369

MGC5509; FLJ45759

Gene Symbol

C22orf28

DDX1

FAM98B

C14orf166

C2orf49

Entrez Gene ID

51493

1653

283742

51637

79074

Table S2. Annotations, domain organization and expression of HSPC117 complex components.

Protein

HSPC117

DDX1

FAM98B

CGI-99

ASW

Gene IDs, Gene Symbols, and aliases were derived from the NCBI-Entrez database (http://www.ncbi.nlm.nih.gov/gene/)

++ Ubiquitously and highly expressed in human tissues, + Ubiquitously expressed in human tissues according to UniGene ESTProfile Viewer (http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=UniGeneAccession)

SOM

Popow et al. 39/44

Table S3. Proteins identified by in solution tryptic digest and MS analysis of affinity

purified c-myc-HSPC117.

Number of Unique Peptides Gene Symbol MW

[kDa] Control Sample


DDX1 82 0 35 1653 DEAD (Asp-Glu-Ala-Asp) box polypeptide 1

C22orf28 55 0 27 51493 chromosome 22 open reading frame 28 FAM98B 46 0 14 283742 family with sequence similarity 98,

member B C14orf166 28 0 12 51637 chromosome 14 open reading frame 166 FAM98A 55 0 10 25940 family with sequence similarity 98,

member A HSPA5 72 0 5 3309 heat shock 70kDa protein 5 RPS14 16 0 3 6208 ribosomal protein S14 RPL11 20 0 3 6135 ribosomal protein L11 KPNA3 56 0 3 3839 karyopherin alpha 3 YWHAE 29 0 2 7531 tyrosine 3-monooxygenase/tryptophan 5-

monooxygenase activation protein, epsilon polypeptide

RPL7 30 0 2 6129 ribosomal protein L7 RPL32P18 16 0 2 644907 ribosomal protein L32 pseudogene 18 RPL10A 25 0 2 4736 ribosomal protein L10a PRSS3 26 0 2 5646 protease, serine, 3 LMNA 78 0 2 4000 lamin A/C

FAM178A 133 0 2 55719 family with sequence similarity 178, member A

DNAH14 519 0 2 127602 dynein, axonemal, heavy chain 14 CSTF3 72 0 2 1479 cleavage stimulation factor, 3' pre-RNA,

subunit 3, 77kDa CSTF1 48 0 2 1477 cleavage stimulation factor, 3' pre-RNA,

subunit 1, 50kDa ATP5B 57 0 2 506 ATP synthase, H+ transporting,

mitochondrial F1 complex, beta polypeptide AHCY 31 0 2 191 adenosylhomocysteinase DDX23 96 3 7 9416 DEAD (Asp-Glu-Ala-Asp) box polypeptide

23 DDX21 80 4 9 9188 DEAD (Asp-Glu-Ala-Asp) box polypeptide

21 RPS3 27 2 4 6188 ribosomal protein S3

RPL21 19 2 4 6144 ribosomal protein L21 MTDH 64 3 5 92140 metadherin RPL28 16 5 8 6158 ribosomal protein L28

TUBA1B 46 4 6 10376 tubulin, alpha 1b RPS18 15 2 3 6222 ribosomal protein S18 RPL24 13 2 3 6152 ribosomal protein L24

EHBP1L1 162 19 26 254102 EH domain binding protein 1-like 1 HSPA8 71 11 15 3312 heat shock 70kDa protein 8 KPNA2 58 6 8 3838 karyopherin alpha 2






RPL34 13 3 4 6164 ribosomal protein L24 RPL3 49 3 4 6122 ribosomal protein L3 RPL15 24 3 4 6138 ribosomal protein L15

HIST1H3D 15 3 4 8351 histone cluster 1, H3d ILF3 96 20 26 3609 interleukin enhancer binding factor 3

TUBB 50 7 9 203068 tubulin, beta TFRC 90 4 5 7037 transferrin receptor ILF2 43 4 5 3608 interleukin enhancer binding factor 2,

45kDa HNRNPA3 37 5 6 220988 heterogeneous nuclear ribonucleoprotein A3

EIF2C2 102 7 8 27161 eukaryotic translation initiation factor 2C, 2 DDX41 70 9 10 51428 DEAD (Asp-Glu-Ala-Asp) box polypeptide

41 HNRNPA2B1 37 12 13 3181 heterogeneous nuclear ribonucleoprotein

A2/B1 KRT1 66 26 28 3848 Keratin 1

HNRNPUL2 85 20 21 221092 heterogeneous nuclear ribonucleoprotein U-like 2

HNRNPU 91 20 21 3192 heterogeneous nuclear ribonucleoprotein U HNRPUL1 96 22 23 11100 Heterogeneous nuclear ribonucleoprotein U-

like 1 PRKDC 459 14 14 5591 protein kinase, DNA-activated, catalytic

polypeptide RPL4 53 5 5 6124 ribosomal protein L4 RPL13 24 5 5 6137 ribosomal protein L13

HNRNPH1 44 5 5 3187 heterogeneous nuclear ribonucleoprotein H1 HIST1H4H 11 5 5 8365 histone cluster 1, H4h

RPS11 18 4 4 6205 ribosomal protein S11 RPL18 19 4 4 6141 ribosomal protein L18 RPL17 21 4 4 6139 ribosomal protein L17

HIST1H2BD 14 4 4 3017 histone cluster 1, H2bd UBC 68 3 3 7316 ubiquitin C

RPS26 12 3 3 6231 ribosomal protein S26 RBBP8 102 3 3 5932 retinoblastoma binding protein 8 EEF1A1 50 3 3 1915 eukaryotic translation elongation factor 1

alpha 1 ZNF326 68 2 2 284695 zinc finger protein 326 YBX1 29 2 2 4904 Y box binding protein 1

RPL37A 8 2 2 6168 ribosomal protein L37a RPL35A 16 2 2 6165 ribosomal protein L35a RPL29 18 2 2 6159 ribosomal protein L29 MYC 49 2 2 4609 v-myc myelocytomatosis viral oncogene

homolog (avian) HIST1H2AB 14 2 2 8335 histone cluster 1, H2ab






TRIM21 54 21 20 6737 tripartite motif-containing 21 LGALS3BP 65 16 14 3959 lectin, galactoside-binding, soluble, 3

binding protein TFG 43 8 7 10342 TRK-fused gene

MATR3 95 8 7 9782 matrin 3 ACTB 41 8 7 60 actin, beta

RBM14 70 15 13 10432 RNA binding motif protein 14 RPL7A 30 7 6 6130 ribosomal protein L7a

HNRNPH3 37 7 6 3189 heterogeneous nuclear ribonucleoprotein H3 FUS 53 7 6 2521 fused in sarcoma

UTP14A 88 13 11 10813 U3 small nucleolar ribonucleoprotein, homolog A (yeast)

VIM 54 6 5 7431 vimentin TNRC6B 187 33 27 23112 trinucleotide repeat containing 6B TNRC6A 171 27 22 27327 trinucleotide repeat containing 6A ZNF280D 127 5 4 54816 zinc finger protein 280D

RPL27 12 5 4 6157 ribosomal protein L27a RPL14 24 5 4 9045 ribosomal protein L14

APOBEC3B 57 8 6 9582 apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B

BBX 68 4 3 56987 bobby sox homolog (Drosophila) HNRNPA1 39 7 5 3178 heterogeneous nuclear ribonucleoprotein A1

BEND3 94 21 14 57673 BEN domain containing 3 RPL8 24 3 2 6132 ribosomal protein L8 RPL27 16 3 2 6155 ribosomal protein L27 NKAP 47 3 2 79576 NFKB activating protein

HIST1H1D 22 3 2 3007 histone cluster 1, H1d CSTA 11 3 2 1475 cystatin A

ARL6IP4 26 3 2 51329 ADP-ribosylation-like factor 6 interacting protein 4

RPS23 16 5 3 6228 ribosomal protein S23 RPS6 29 7 4 6194 ribosomal protein S6 RPL6 33 6 3 6128 ribosomal protein L6 RPL23 15 4 2 9349 ribosomal protein L23 RPL10 25 4 2 6134 ribosomal protein L10 DSP 332 11 5 1832 desmoplakin

HNRNPAB 36 6 2 3182 heterogeneous nuclear ribonucleoprotein A/B

JUP 82 10 3 3728 junction plakoglobin IGH@ 51 7 2 3492 immunoglobulin heavy locus

ZNF192 66 4 0 7745 zinc finger protein 192 VCP 89 3 0 7415 valosin-containing protein TF 77 2 0 7018 transferrin

TAF15 63 3 0 8148 TATA box binding protein (TBP)-associated factor, 68kDa






SMC3 142 9 0 9126 structural maintenance of chromosomes 3 SMC1A 143 5 0 8243 structural maintenance of chromosomes 1A SBSN 61 2 0 374897 suprabasin RPS9 17 2 0 6203 ribosomal protein S9

RPS4X 43 8 0 6191 ribosomal protein S4, X-linked RPS13 17 3 0 6207 ribosomal protein S13 RPL36 12 2 0 25873 ribosomal protein L36 RPL19 23 2 0 6143 ribosomal protein L19

RPL18A 21 2 0 6142 ribosomal protein L18a RP9 26 4 0 6100 retinitis pigmentosa 9 (autosomal dominant) RIF1 257 7 0 55183 RAP1 interacting factor homolog (yeast)

RAD21 66 2 0 5885 RAD21 homolog (S. pombe) PCMT1 25 2 0 5110 protein-L-isoaspartate (D-aspartate) O-

methyltransferase PABPC1 47 2 0 26986 poly(A) binding protein, cytoplasmic 1 MUCL1 9 2 0 118430 mucin-like 1

IgM 57 2 0 n.a. Immunoglobulin M IGHA1 54 3 0 3493 immunoglobulin heavy constant alpha 1

IgG light chain

12 2 0 n.a. Immunoglobulin G, light chain

IgA light chain

23 3 0 n.a. Immunoglobulin A, light chain

Ig kappa chain

23 3 0 n.a. immunoglobulin kappa-chain VK-1

HNRNPM 78 3 0 4670 heterogeneous nuclear ribonucleoprotein M GAPDH 36 2 0 2597 glyceraldehyde-3-phosphate dehydrogenase

FLG2 248 3 0 388698 filaggrin family member 2 FLG 123 6 0 2312 filaggrin

FAM133B 22 2 0 257415 family with sequence similarity 133, member B

DSG1 114 5 0 1828 desmoglein 1 CIR1 50 2 0 9541 corepressor interacting with RBPJ, 1

C5 188 2 0 727 complement component 5 C3 187 6 0 718 complement component 3

AZGP1 32 2 0 563 alpha-2-glycoprotein 1, zinc-binding APOD 20 2 0 347 apolipoprotein D A2M 163 3 0 2 alpha-2-macroglobulin


Supplemental references

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S2. P. P. Chan, T. M. Lowe, Nucleic Acids Res 37, D93 (Jan, 2009).

S3. G. D. Tocchini-Valentini, P. Fruscoloni, G. P. Tocchini-Valentini, Proc Natl Acad Sci U S A 102, 8933 (Jun 21, 2005).

S4. M. Englert, H. Beier, Nucleic Acids Res 33, 388 (2005).

S5. S. F. Altschul et al., Nucleic Acids Res 25, 3389 (Sep 1, 1997).

S6. K. Katoh, H. Toh, Brief Bioinform 9, 286 (Jul, 2008).

S7. J. D. Thompson, T. J. Gibson, D. G. Higgins, Curr Protoc Bioinformatics Chapter 2, Unit 2 3 (Aug, 2002).

S8. C. L. Will et al., Embo J 20, 4536 (Aug 15, 2001).

S9. N. Bouzaidi-Tiali, E. Aeby, F. Charriere, M. Pusnik, A. Schneider, Embo J 26, 4302 (Oct 17, 2007).

S10. M. van de Wetering et al., EMBO Rep 4, 609 (Jun, 2003).

S11. R. Kittler et al., Proc Natl Acad Sci U S A 102, 2396 (Feb 15, 2005).

S12. V. Drewett et al., Nucleic Acids Res 29, 479 (Jan 15, 2001).

S13. C. Okada, Y. Maegawa, M. Yao, I. Tanaka, Proteins 63, 1119 (Jun 1, 2006).

S14. M. Englert, K. Sheppard, A. Aslanian, J. Yates, D. Söll, Proc Natl Acad Sci U S A, in press.

S15. C. L. de Hoog, L. J. Foster, M. Mann, Cell 117, 649 (May 28, 2004).

S16. J. Hu et al., Front Biosci 13, 7123 (2008).

S17. Y. Wang et al., Biochem Biophys Res Commun 397, 407 (2010).

S18. S. L. Howng et al., FEBS Lett 566, 162 (May 21, 2004).

S19. M. Y. Galperin, E. V. Koonin, Nucleic Acids Res 32, 5452 (2004).

S20. O. Cordin, J. Banroques, N. K. Tanner, P. Linder, Gene 367, 17 (Feb 15, 2006).

S21. R. D. Finn et al., Nucleic Acids Res 38, D211 (Jan, 2010).


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