Chapter 6

RNA post-transcriptional

processing

RNA Processing• Very few RNA molecules are transcribed

directly into the final mature RNA.• Most newly transcribed RNA molecules

(primary transcripts) undergo various alterations to yield the mature product

• RNA processing is the collective term used to describe the molecular events allowing the primary transcripts to become the mature RNA.

primary transcriptprimary transcript

mature RNAmature RNA..

Nucleus or NucleolusCytoplasm

RNA

processing

Romoval of nucleotidesaddition of nucleotides to the 5’- or 3’- ends

modification of certain nucleotides

(1) Removal of nucleotides by both endonucleases and exonucleases endonucleasesendonucleases to cut at specific to cut at specific

sites sites withinwithin a precursor RNAa precursor RNA exonucleasesexonucleases to trim the to trim the endsends of a of a

precursor RNAprecursor RNA This general process is seen in This general process is seen in

prokaryotes and eukaryotes for all prokaryotes and eukaryotes for all types of RNAtypes of RNA

(2) Addition of nucleotides to 5’-or 3’-ends of the primary transcripts or their cleavage products.

Add a cap and a Add a cap and a poly(A)poly(A) tail to pre- tail to pre-mRNAmRNA

(3) Modification of certain nucleotides on either the base or the sugar moiety.

–Add a methyl group to 2’-OH of ribose in mRNA (A) and rRNA

–Extensive changes of bases in tRNA

RNPs( 核糖核蛋白 )Ribonucleoproteins =

RNA protein complexes The RNA molecules in cells usually The RNA molecules in cells usually

exist complexed with proteinsexist complexed with proteins specific proteins attach to specific specific proteins attach to specific

RNAsRNAs RibosomesRibosomes are the largest and most are the largest and most

complex RNPscomplex RNPs

3-D structure

Digital cryo-electron micrographyRNP

颗粒的低温电镜

图

6.1: rRNA PROCESSING

• rRNA processing in prokaryotes• rRNA processing in eukaryotes

6.1-1: rRNA processing in prokaryotes1. There are 7 different operons for rRNA

that are dispersed throughout the genome.

2. Each operon contains one copy of each of the 5S,the 16S and the 23S rRNA sequences. About 1~4 coding sequences for tRNA molecules are also present in these rRNA operons.

3. The initial transcript has a sedimentation coefficient of 30s (6000 nt) and is normally quite short-lived

rRNA operon

Pre-16S rRNA Pre-tRNA Pre-23S rRNA pre-5S rRNA

Pre-tRNA

Promoters Terminators

• RNase III, involved in the first step of rRNA processing

• RNase M5, M16 and M23 are involved in the second step of rRNA processing

30S pre-rRNA

Transcription

Pre-16S rRNA Pre-tRNA Pre-23S rRNA pre-5S rRNA

Pre-tRNA

Promoters Terminators

Processing steps

Step 1: Following or during the primary transcription, the RNA folds up into a number of stem-loop structures by base pairing between complementary sequences

RNA folding

Step 2: The formation of this secondary structure of stems and loops allows some proteins to bind to form a RNP complex which remain attached to the RNA and become part of the ribosome

RNP complex formation

Step 3: After the binding of proteins, nucleotide modifications take place.

Example: methylation of adenine by methylating agent S-Adenosylmethonine (SAM)

Step 4: RNA cleavage

Pre-16S rRNA Pre-tRNA Pre-23S rRNA pre-5S rRNA Pre-tRNA

Promoters Terminators

30S pre-rRNA: Transcription

Cleavage at

16S rRNA tRNA 23S rRNA 5S rRNA tRNA

RNase III III P F III III P F P ERNase III III P F III III P F P E

RNase M16 M16 M23 M23 M5RNase M16 M16 M23 M23 M5

rRNA operon

6.1-2: rRNA processing in eukaryotes• rRNA in eukaryotes is also

generated from a single, long precursor molecule by specific modification and cleavage steps

• The processes are not so well understood

1. The rRNA genes are present in a tandemly repeated cluster containing 100 or more copies of the transcription unit, and are transcribed in nucleolus by RNA Pol I

2. Precursor sizes are different among organisms (yeast: 7000 nt; mammalian 13500 nt), and pre-mRNA processing is also slightly different among organism.

3. The precursor contains • one copy of the 18S coding region and • one copy each of the 5.8S and 28S coding

regions, which together are the equivalent of the 23S rRNA in prokaryote

4. The large precursor RNA undergoes a number of cleavages to yield mature RNA and ribosome.

5. The eukaryotic 5S rRNA • is transcribed by RNA Pol III from

unlinked genes to give a 121nt transcript • the transcript undergoes little or no

processing

rRNA 前体加工的基本步骤① 在转录的 45S 前体 rRNA 上发生甲基化作用；② 在 5’ 端切除非编码序列生成 41S rRNA ；③41S rRNA 再被切割成两段，一段为 32S ，含有 28S 和 5.8S rRNA ，另一段为 20S ，含有 18S rRNA ；④32S 被剪切成 28S 和 5.8S rRNA ， 28S 和5.8S rRNA 中的部分序列互补，配对；⑤20S 被剪切成 18S rRNA ；

18S rRNA 5.8S rRNA 28S rRNA

45S

41S

20S and 32S

Mature rRNAs

47S18S 5.8S 28S

ETS1 ITS1 ITS2 ETS2

Indicates RNase cleavageMammalian pre-rRNA processing

• The 5.8S region must base-pair to the 28S rRNA before the mature molecules are produced.

• Mature rRNAs complex with protein to form RNPs (nucleolus)

• Methylation occurs at over 100 sites to give 2’-O-methylribose, which is known to be carried out by snRNPs (nucleolus)

• Introns (group I) in rRNA genes of some lower eukarytes (Tetrahymena thermophila) must be spliced out to generate mature rRNAs.

• Many group I introns are found to catalyze the splicing reaction by itself in vitro, therefore called ribozyme

6.2: tRNA PROCESSING, RNase P AND RIBOZYMES • tRNA processing in prokaryotes• tRNA processing in eukaryotes• RNase P• Ribozymes

tRNA 3-D structure

tRNA processing in prokaryotes

Mature tRNAs are generated by processing longer pre-tRNA transcripts, which involves

1. specific exo- and endonucleolytic cleavage by RNases D, E, F and P (general) followed by

2. base modifications which are unique to each particular tRNA type.

Primary transcriptsRNase D,E,F and P

(See your text book) tRNA with mature ends

Base modifications

mature tRNAs

tRNA processing in eukaryotes

The pre-tRNA is synthesized with a

1. 16 nt 5’-leader, 2. a 14 nt intron and 3. two extra 3’-nucleotides.

1. Primary transcripts forms secondary structures recognized by endonucleases

2. 5’ leader and 3’ extra nucleotide removal3. tRNA nucleptidyl transferase adds 5’-CC

A-3’ to the 3’-end to generate the mature 3’-end

4. Intron removal

tRNA 加工修饰过程包括：①RNA 内切核酸酶在 tRNA5’ 端切断，使 5’端逐步成熟；②RNA 内切核酸酶在 tRNA3’ 端切断，再由

RNA 外切核酸酶从 3’ 端逐个切去附加序列；③ 在 tRNA3’ 端添加－ CCAOH ；④ 核酸的修饰和异构化。

1 由 RNaseP 切割成 tRNA 片段，但是 3’ 和 5’ 端仍需要修饰；2 RNaseF 从 3’ 端切割，由 RNaseD 外切酶对 3’ 端进一步加工， RNaseD 是 3’ 端的成熟酶；3 在 tRNA 核苷酰转移酶的作用下，添加 3’ 端 CCAOH

4 tRNA 分子中存在许多修饰碱基，包括甲基化修饰，假尿嘧啶 修饰等。

1 、 tRNA 3’ 端的成熟

2 、 tRNA 5’ 端的成熟 RNaseIII 切割 tRNA 的片段中， 5’ 端含有多余的核苷酸，通过 RNaseP 切除。因此， RNaseP 是 t

RNA 5’ 端的成熟酶。

RNase P• Ribonuclease P (RNase P) is an enzyme

involved in tRNA processing that removes the 5' leader sequences from tRNA precursors

RNase P (2)• RNase P enzymes are found in both prok

aryotes and eukaryotes, being located in the nucleus of the latter where they are therefore small nuclear RNPs (snRNPs)

• In E. coli, the endonuclease is composed of a 377 nt RNA and a small basic protein of 13.7kDa.

RNase P (1)• RNA component can catalyze pre-tRNA

in vitro in the absence of protein. Thus RNase P RNA is a catalytic RNA, or ribozyme.

Ribozyme (1)• Ribozymes are catalytic RNA molecules t

hat can catalyze particular biochemical reactions.

• RNase P RNA is a ribozyme.• RNase P RNA from bacteria is more catal

ytically active in vitro than those from eukaryotic and archaebacterial cells. All RNase P RNAs share common sequences and structures.

Ribozyme (2) Self-splicing introns: the intervening RNA that catalyze the splicing of themselves from their precursor RNA, and the joining of the exon sequences1. Group I introns, such as Tetrahymena intron2. Group II introns.

Ribozyme (3) Self-cleaving RNA encoded by viral genome to resolve the concatameric molecules of the viral genomic RNA1. HDV ribozyme2. Hairpin ribozyme3. Hammer head ribozyme

Ribozyme (4)Ribozymes can be used as

therapeutic agents in 1. correcting mutant mRNA in human

cells2. inhibiting unwanted gene

expression Kill cancer cells Prevent virus replication

6.3: mRNA PROCESSING, hnRNPs AND snRNPs• Processing of mRNA• hnRNP• snRNP particles• 5’Capping• 3’Cleavage and polyadenylation• Splicing• Pre-mRNA methylation

Processing of mRNA: prokaryotes

• There is essentially no processing of prokaryotic mRNA, it can start to be translated before it has finished being transcribed.

• Prokaryotic mRNA is degraded rapidly from the 5’ end

Processing of mRNA in eukaryotes

• In eukaryotes, mRNA is synthesized by RNA Pol II as longer precursors (pre-mRNA), the population of different RNA Pol II transcripts are called heterogeneous nuclear RNA (hnRNA).

• Among hnRNA, those processed to give mature mRNAs are called pre-mRNAs

Pre-mRNA molecules are processed to mature mRNAs by 5’-capping, 3’-cleavage and polyadenylation, splicing and methylation.

Eukaryotic mRNA processing: overview

hnRNP: hnRNA + proteins• The hnRNA synthesized by RNA Pol II is

mainly pre-mRNA and rapidly becomes covered by proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP)

• The hnRNP proteins are though to help keep the hnRNA in a single-stranded form and to assist in the various RNA processing reactions

snRNP particles: snRNA + proteins1. snRNAs are rich in the base uracil, whi

ch complex with specific proteins to form snRNPs.

2. The most abundant snRNP are involved in pre-mRNA splicing, U1,U2,U4,U5 and U6.

3. A large number of snRNP define methylation sites in pre-rRNA.

• snRNAs are synthesized in the nucleus by RNA Pol II and have a normal 5’-cap.

• Exported to the cytoplasm where they associate with the common core proteins and with other specific proteins.

• Their 5’-cap gains two methyl groups and then imported back into the nucleus where they function in splicing.

5’ Capping• Very soon after RNA Pol II starts makin

g a transcript, and before the RNA chain is more then 20 -30 nt long, the 5’-end is chemically modified.

• 7-methylguanosine is covalently to the 5´ end of pre-mRNA.

• Linked 5´ 5´• Occurs shortly after initiation

7-methylguanosine (m7G)

Function of 5´cap

• Protection from degradation• Increased translational efficiency• Transport to cytoplasm• Splicing of first exon

3’ Cleavage and polyadenylation

• In most pre-mRNAs, the mature 3’-end of the molecule is generated by cleavage followed by the addition of a run, or tail, of A residues which is called the poly(A) tail.

• RNA polymerase II does not usually terminate at distinct site

• Pre-mRNA is cleaved ~20 nucleotides downstream of polyadenylation signal (AAUAAA)

• ~250 AMPs are then added to the 3´ end

• Almost all mRNAs have poly(A) tail

Function of poly(A) tail

• Increased mRNA stability• Increased translational efficiency• Splicing of last intron

Splicing• the process of cutting the pre-mRNA to r

emove the introns and joining together of the exons is called splicing.

• it takes place in the nucleus before the mature mRNA can be exported to the cytoplasm.

• Introns: non-coding sequences• Exons: coding sequences• RNA splicing: removal of introns and

joining of exons• Splicing mechanism must be precise to

maintain open reading frame• Catalyzed by spliceosome 剪接体 (RNA

+ protein)

Biochemical steps of pre-mRNA splicing

Step 1: a cut is made at the 5′splice site, separating the left exon and the right intron-exon molecule. The right intron-exon molecule forms a lariat, in which the 5′terminus of the intron becomes linked by a 5′-2′ bond to a base within the intron. The target base is an A in a sequence that is called the branch site

Step 2: cutting at the 3′ splice site releases the free intron in lariat form, while the right exon is ligated (spliced) to the left exon.

C U R A YLariat

Nuclear splicing occurs by two transesterification reactions in which a free OH end attacks a phosphodiester bond.

Spliceosome• Catalyzes pre-mRNA splicing in nucleus• Composed of five snRNPs (U1, U2, U4, U5 and

U6), other splicing factors, and the pre-mRNA being assembled

• U1 binds to the 5’ splice site, then U2 to the branchpoint, then the tri-snRNP complex of U4, U5 and U6. As a result, the intron is looped out and the 5’- and 3’ exon are brought into close proximity.

• U2 and U6 snRNA are able to catalyze the splicing reaction.

Splicing cycle

U1 snRNA splicing

② U2 snRNP

U2 snRNP 能够与内含子的分支点处序列互补，此外还与 U6 snRNA 碱基配对。在高等真核生物中 U2 snRNP 与内含子的结合还需要 U2AF （ U2 associated factor or Auxiliary factor) 的协助。

③U4 snRNP U4 snRNP 与 U6 snRNP 偶联，当剪接体组装后，需要 U6 snRNP 参与剪接时， U4 snRNP 被释放出来。④U5 snRNP U5 snRNP 与其它 snRNA 没有明显的作用，但它与 pre-mRNA的 5‘’和 3‘’端剪切位点又相互作用。⑤U6 snRNP U6 snRNP 与内含子 5’端剪接区序列配对。酵母 U6 snRNP 的保守序列为 ACAGAG 。可能结合模式为 5’端剪切区 47－ 49 位与 pre-mRNA 的内含子第 4－ 6位的 UGU 结合；5’端剪切区 42－ 44 位的 ACA 与 UGU 结合。

GUAUGU

GAG ACAUAACAAAGUU6 snRNA

5’

3’5’

5248 43 38

GUAUGU

GAGACAUA ACAAAGUU6 snRNA

5’

3’5’

52 48 43 38

U1

Cap2

U6

U2

U5

Intron

5‘E1

E2 3’

A

U6/U4U6/U2

• SnRNA U1 ~ 5’, 3’ consensus seq

• SnRNA U2 ~ Branch Site ( 其中 A 为转酯攻击位点 )

• SnRNA U6 ~ 5’ consensus seq.

• SnRNA U5 与 5’~GU & 3’~AG 配对，以连接 exons

• SnRNA U6 ~ U4 SnRNA U6 ~ U2 稳定 spliceosome

• Set of SR protein (rich Ser & Arg)

5’ of Intron 剪切与 lariat 的形成同步 3’ of intron 剪切与 lariat 切除， exons 连接同步

spliceosome 解体与 lariat 降解同步

Cut-ligate

Pre-mRNA methylation• The final modification or processing

event that many pre-mRNAs undergo is specific methylation of certain bases.

• The methylations seem to be largely conserved in the mature mRNA.

6.4: ALTERNATIVE mRNA PROCESSING

• Alternative processing

• Alternative poly(A) sites

• Alternative splicing

• RNA editing

Alternative processing Alternative mRNA processing is the

conversion of pre-mRNA species into more than one type of mature mRNA.

Types of alternative RNA processing include alternative (or differential) splicing and alternative (or differential) poly(A) processing.

Alternative poly(A) sites• Some pre-mRNAs contain more than

one poly(A) site and these may be used under different circumstances to generate different mature mRNAs.

• In one cell the stronger poly(A) site is used by default, but in other cell a factor may prevent stronger site from being used.

Alternative splicing• The generation of different mature mRN

As from a particular type of gene transcript can occur by varying the use of 5’- and 3’- splice sites in four ways:

(i) By using different promoters(ii) By using different poly(A) sites(iii) By retaining certain introns(iv) By retaining or removing certain exons

Alternative splicing

Alternative splicing

(A) A cassette exon can be either included in the mRNA or excluded.

(B) Mutually exclusive exons occur when two or more adjacent cassette exons are spliced such that only one exon in the group is included at a time.

(C, D) Alternative 5’ and 3’ splice sites allow the lengthening or shortening of a particular exon.

(E, F) Alternative promoters and alternative poly(A) sites switch the 59- or 39-most exons of a transcript.

(G) A retained intron can be excised from the pre-mRNA or can be retained in the translated mRNA.

(H) A single pre-mRNA can exhibit multiple sites of alternative splicing using different patterns of inclusion.

Sex in Drosophila is largely

determined by alternative

splicing

Sxl-Sex lethal gene: the master regulatory gene at the top of the sex determination pathway. The downstream targets of the Sxl protein include transcripts from the Transformer (Tra) and Male-specific lethal 2 (Msl2) genes. Tra gene also encodes a splicing regulator.

RNA editing

• An unusual form of RNA processing in which the sequence of the primary transcript is altered is called RNA editing.

• Changing RNA sequence (after transcription)

RNA editing is known to occur in two different situations, with different causes. In mammalian cells there are cases in which a substitution occurs in an individual base in mRNA, causing a change in the sequence of the protein that is coded. (Base modification:A or C deamination) In trypanosome mitochondria, more widespread changes occur in transcripts of several genes, when bases are systematically added or deleted. (Base U insertion and deletion)

1. Apolipoprotein-B mRNA in mammalian intestine and liver. The genome contains a single (interrupted) gene whose sequence is identical in all tissues, with a coding region of 4563 codons.

• a protein of 512 kD representing the full coding sequence is found in the liver.

• A shorter form of the protein, ~250 kD, is synthesized in intestine. This protein consists of the N-terminal half of the full-length protein, which is caused by the change of a CAA codon to a UAA. (see Figure)

Deamination in mammalian

Liver Intestine

Deamination in mammalian2. glutamate receptors in rat brain. • Editing at one position changes a glutamine codon in DNA into a codon for arginine in RNA (AI)• The change affects the conductivity of the channel and therefore has an important effect on controlling ion flow through the neurotransmitter.

Deamination is catalyzed by cytidine or adenosine deaminases.Editing enzymes are related to the general deaminases, but have other regions or additional subunits that control their specificity.

Insertion or deletion of U in protozoa

Example: part of the mRNA sequence of Trypanosome brucei coxIII shows many uridines that are not coded in the DNA (shown in red) or that are removed from the RNA (shown as T).

Insertion or deletion of U in protozoa

A guide RNA containing a sequence that is complementary to the correctly edited mRNA provides a mechanism of U insertion or deletion. (See figure)