Review Article Nuclear Distribution of RNA Polymerase II and...

Review ArticleNuclear Distribution of RNA Polymerase II and mRNAProcessing Machinery in Early Mammalian Embryos

Irina O Bogolyubova and Dmitry S Bogolyubov

Laboratory of Cell Morphology Institute of Cytology RAS 4 Tikhoretsky Avenue St Petersburg 194064 Russia

Correspondence should be addressed to Irina O Bogolyubova ibogolmailru

Received 27 February 2014 Accepted 11 April 2014 Published 29 April 2014

Academic Editor Hiromichi Matsumoto

Copyright copy 2014 I O Bogolyubova and D S BogolyubovThis is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Spatial distribution of components of nuclear metabolism provides a significant impact on regulation of the processes of geneexpression While distribution of the key nuclear antigens and their association with the defined nuclear domains were thoroughlytraced in mammalian somatic cells similar data for the preimplantation embryos are scanty and fragmental However the periodof cleavage is characterized by the most drastic and dynamic nuclear reorganizations accompanying zygotic gene activation Inthis minireview we try to summarize the results of studies concerning distribution of major factors involved in RNA polymeraseII-dependent transcription pre-mRNA splicing mRNA export that have been carried out on early embryos of mammals

1 Introduction

The main feature of the eukaryotic cell is the nucleus thatholds the genetic information and realizes DNA replica-tion and the processes of gene expression Being the placewhere transcription and the related events occur the nucleusdemarcates nuclear processes from translation in cytoplasmproviding new additional levels of the regulation of geneexpression that cannot be realized in prokaryotes

Interchromatin space of the nucleus contains differentnuclear domains In mammalian somatic cells molecularcomposition of major functional nuclear domains has beenstudied in detail [1] Initially the nuclear distribution of pre-mRNA splicing factors and the structures enrichedwith thesefactors have been explored with the use of antibodies againstthe Sm antigen (now known as symmetric dimethylargininesDMA) [2] 227-trimethylguanosine (TMG) cap of snRNAsand some ldquosignaturerdquo proteins of nuclear domains (coilinSR-proteins) [3] As a result interchromatin granule clusters(IGCs) known as nuclear splicing speckles in terms offluorescent microscopy and also Cajal bodies have attracteda special attention as evolutionary conserved and ldquouniversalrdquonuclear domains [4]

IGCsspeckles are nuclear domains enriched in pre-mRNA splicing factors (snRNPs and SR-proteins) locatedin the interchromatin space of the nucleus These domainsare dynamic nuclear organelles and their constituents canexchange continuously with the nucleoplasm and othernuclear locations including active transcription sites [5]IGCs not only serve as transient reservoirs for pre-mRNAsplicing factors but are also involved inmany other functions(for reviews see [5 6])TheCajal bodies have been implicatedin RNA-related metabolic processes such as snRNP biogene-sis maturation and recycling histonemRNAprocessing andtelomere maintenance (for reviews see [7 8]) Decipheringthe fundamental rules that impact nuclear functions inthe whole seems to be impossible without a large-scalecomparative approach As compared with traditionally usedexperimental objects including tissue-culture somatic cellsearly mammalian embryos have been explored with a lesserextent

Thenuclei of earlymammalian embryos are characterizednot only by the unique functional status but also by apeculiar nuclear ultrastructure [9] Stepwise physiologicalreactivation of chromosomal transcription activity duringearly embryogenesis known in literature as zygotic gene acti-vation (ZGA) suggests that the embryos could be attractive

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 681596 9 pageshttpdxdoiorg1011552014681596

2 BioMed Research International

experimental models in order to analyze nuclear distributionof gene expression factors that virtually may associate withthe universal andor specific nuclear domains ZGA is one ofthe key points of the maternal-to-zygotic transition (MZT)that involves a set of structural molecular and biochemicalrearrangements [10ndash14] However the majority of studiesconcerned first of all the changes of gene expression patterns[15ndash17] as well as the processes of chromatin remodelingduring realization of ZGA events (for reviews see [18 19])The relationships between these processes and the distribu-tion of the essential components of nuclear metabolism weredescribed with a lesser extent

Here we review available literary data on nuclear distribu-tion of some essential molecular components related to RNApolymerase II-dependent transcription and mRNA process-ing at different stages of ZGA in mammals The dynamicsof the nucleolar components in mammalian embryos wasstudied better [20ndash22] and remains beyond the scope of thepresent review

2 RNA Polymerase II and BasalTranscription Factors

The association of RNA polymerase II (RNAP II) with manyfactors to form a polyfunctional holoenzyme is requiredfor transcription [47 48] Besides core RNAP II enzymethis complex includes basal transcription factors mediatorcomplexes and transcription coactivators [49ndash51] The largeRNAP II subunit (RPB1) contains the carboxy-terminaldomain (CTD) consisting of multiple heptapeptide repeatswith the consensus sequence YSPTSPS Dynamic phospho-rylation of the CTD determines the progression of the RNAPII transcription cycle from initiation through elongation totermination [52ndash54]

Posttranslational modifications of RNAP II holoenzymewere studied in mouse and rabbit preimplantation embryoswith the use of western blot analysis and immunofluores-cent microscopy [23] In this study a set of the followingmonoclonal antibodies was applied 8WG16 revealing theunphosphorylated CTD CC-3 directed against a phospho-rylated epitope of the CTD and Pol33 against an internalepitope distinct from the CTD thus recognizing two forms ofRPB1 simultaneously The authors have eventually suggestedthat phosphorylation of the CTD might control ZGA inthe embryos of mammals with different ZGA chronologiesSeveral hours after fertilization the CTD was found dephos-phorylated Dephosphorylation of the CTD occurs before theonset of a period characterized by a weak transcriptionalactivity (minor ZGA) Then the CTD lacks immunologicaland drug-sensitivity characteristics related to its phospho-rylation status and RNAP II gradually translocates intothe nuclei At the major ZGA phase corresponding the2-cell stage in mouse and the 8ndash16-cell stage in rabbitphosphorylation pattern of the CTD was close to thatobserved in somatic cells The authors also reported thatactinomycin D does not prevent a new phosphorylationpattern of RNAP II at the onset of the major ZGA Asa result a phosphorylated embryogenesis-specific RNAP II

isoform was identified This RNAP II isoform is insensitiveto DRB a CTD-kinase inhibitor since it lacks a phosphoepi-tope generated by TFIIH-associated kinase phosphorylationFinally the authors have concluded that nuclear translocationof RNAP II and CTD phosphorylation might be majordeterminants of ZGA The possible association of differentRNAP II isoforms with intranuclear structures of mouse andrabbit preimplantation embryos was not explored in detail inthis study

In our immunocytochemical experiments we used anaffinity purified polyclonal serum to reveal the hyperphos-phorylated form of RNAP II in mouse embryos This formof RNAP II was detected before the major ZGA phasenamely in early 2-cell embryos [24] While ZGA proceedsRNAP II was found in association with perichromatin fibrils(PFs) [25] which are referred to as the ultrastructural ldquoinsitu formsrdquo of nascent pre-mRNA transcripts [55] Besidesthe hyperphosphorylated RNAP II was detected in nuclearspeckles identified by the presence of the SR protein SC35The intensity of anti-RNAP II immunostaining in speckleswas being increased towards the end of ZGA [24] (Figure 1)If ZGA is delayed for example in embryos under the so-called ldquo2-cell block in vitrordquo (for details see [56 57]) thehyperphosphorylated form of RNAP II begins to accumulatein enlarged nuclear speckles [26] (Figure 2)

Worrad et al [29] have explored the concentration of thetranscription factors Sp1 and TBP (TATA-binding subunit ofTFIID) in 1-cell mouse embryos The authors showed thatconcentration of both factors drastically increases in a time-dependent fashion after fertilization during the 1-cell stageSix hours following the formation of pronuclei this increasecontinued and by the G2 phase the pronuclear concentrationof Sp1 and TBP was very similar to that observed in 2-cellembryos In addition concentration of both transcriptionfactors was greater in the male pronucleus and the differencewas more pronounced for TBP

Later TBP and TBP-associated factor 1 (TAF1) wererevealed in mouse zygotes [30] TAF1 was not detectedjust after fertilization in transcriptionally silent cells butit is expressed in pronuclei reaching the maximum beforethe onset of ZGA TAF1 and TBP shared similar dynamicsof expression patterns In 4 h after fertilization anti-TAF1immunofluorescent signal was not registered The signalbecame obvious in a portion of embryos in 6 h after fertil-ization in the majority of embryos in 9 h and in all embryosin 11 h TBP was revealed in some embryos for the first timein 4 h after fertilization and in almost all pronuclei 1 h laterIn male pronuclei transcriptional activity can be registeredearlier Respectively both factors initially appear in malerather than in female pronuclei The authors have supposedthat the deficiency of transcription machinery might be areason for the limitation of transcription at the beginning ofembryogenesis

In addition to TBP Gazdag et al [31] have revealedthe related protein TBP2 also called TRF3 (TBP-relatedfactor 3) in mouse 1-2-cell embryos They also reportedthat TBP is expressed in the oocytes at the beginning offolliculogenesis but cannot be detected during further stagesof oocyte development and becomes abundant again only

BioMed Research International 3

32h SC35 Merge

(a) (a998400) (a998400998400)

(b) (b998400) (b998400998400)

46h

PolII

Figure 1 Double immunolocalization of SC35 (a) (b) and hyperphosphorylated RNA polymerase II (PolII) (a1015840) (b1015840) in mouse embryosFluorescence of nuclei begins to be detected only at the early 2-cell stage (line a) Association of RNAP II with SC35 domains (speckles) isobserved already at this stage and is increased when ZGA finishes (line b) Bar is 10 120583m according to [24] open access

46 in vitro SC35

(a)

PolII

(b)

72 in vitro

(c) (d)

Figure 2The distribution of SR protein SC35 (a) (c) hyperphosphorylated RNApolymerase II (PolII) (b) (d) in control (a) (b) and arrestedin vitro 2-cell mouse embryos (c) (d) Note the large SC35 speckles enriched in hyperphosphorylated RNA polymerase II in the nucleus ofblocked embryo Scale bar is 5120583m according to [26] reprinted from Tissue and Cell Bogolyubova Transcriptional activity of nuclei in 2-cellblocked mouse embryos 2011 43 262-265 [26] with permission from Elsevier


Table 1 RNA polymerase II mRNA processing factors and mRNA export factors revealed in early mammalian embryos

Group of factors Animal Detected antigen Method Stage of embryogenesis Reference

RNAP II Mouse rabbit core of the RPB1RNAPIIa RNAPIIo IFlowast WB 1-cellndash16ndash32 cell [23]

Mouse RNAPIIa RNAPIIo IF IEM 1-cell-2-cell [24ndash27]

mRNA transcriptionfactors Mouse

TBP IF 1-cell [28]

Sp1 TBP

IFMicroinjection of

Sp1-dependent luciferasereporter gene

1-cell-2-cell [29]

TBP TAF1 IF 1-cell [30]TBP TBP2 IF WB 1-cell-2-cell [31]TFIID IF 1-cell-2-cell [24]

Splicing factors

Mouse

U1 U2 U4 U6 snRNAssnRNPs

ISHIF 1-cell-blastocyst [32]

U1 snRNAU1 snRNP


U1 U2 snRNAssnRNPs SC35

ISHIF 1-cellndash8-cell [34]

snRNPs SC35 IF IEM 1 cell-2-cell [24 35 36]snRNPs IF 1-cell-2-cell [27]snRNPs IEM 2-cell [37]hnRNPs snRNPs IEM 1-cellndash8-cell [38]

BovineU2 snRNAsnRNPs

ISH NBIF 1-cell-blastocyst [39]

snRNPs SC35 IEM 1-cellndash16-cell [40 41]Caprine snRNPs SC35 IEM 1-cell-2-cell [41]

PorcinesnRNPs IF 1-cell-blastocyst [42]snRNPs SC35 IEM 1-cellndash4-cell [43]

HamsterU1 U2 snRNAssnRNPsSC35

ISHIF

IF IEM1-cellndash8-cell [44]

mRNA export-relatedfactors

Mouse hnRNPs IF WB 1-cellndash8-cell1-cell

[34][28]

hnRNPs Y14 AlyREFNXF1TAP IF IEM 1-cell-2-cell [45 46]

Hamster hnRNPs IF 1-cellndash8-cell [44]lowastAbbreviations IF immunofluorescence IEM immunoelectronmicroscopy ISH in situ hybridizationNB northern blotting RNAPIIa hypophosphorylatedform of RNA polymerase II RNAPIIo hyperphosphorylated form of RNA polymerase II WB western blotting

after fertilization In contrast to TBP TBP2 was almostundetectable after fertilization until the 2-cell stage

In mouse embryo pronuclei basal transcription factorTFIID has been revealed before the onset of ZGA in associ-ation with nuclear speckles already at the zygote stage [24]During realization of ZGA the presence of TFIID in thespeckles became more evident (Figure 3) Localization dataof RNAP II and TFIID in the IGCs of mouse embryos arein accordance with the results that came from somatic cellstudies [58 59] including IGC proteome analysis [60]

Interestingly TFIID was also clearly detectable at theperiphery of the nucleolar precursor bodies (NPBs) at theearliest stages of mouse cleavage [24] The functional sig-nificance of this finding is ambiguous Further studies are

required to clarify the molecular composition and functionsof NPBs enigmatic structures of embryo nuclei

Synoptic data on the revealing of RNAP II and transcrip-tion factors in mammalian embryos are presented in Table 1At least a portion of RNAP II transcription machinery isdetected in mammalian embryo nuclei already before ZGAHowever the final intranuclear patterns of RNAP II andtranscription factors are being formed for the whole periodof ZGA

3 Pre-mRNA Splicing Factors

Pre-mRNA splicing factors have been detected in theembryos of various mammalian species (Table 1) In the


(a) (a998400) (a998400998400)

(b) (b998400) (b998400998400)

24h SC35 Merge

46h

TFIID

Figure 3 Double immunolocalization of SC35 (a) (b) and transcription factor TFIID (a1015840) (b1015840) in mouse embryos TFIID is revealed in thenuclei at all studied stages Colocalization of SC35 and TFIID is intensified during ZGA Bar is 10 120583m according to [24] open access

nuclei of early and late 2-cell mouse embryos the distributionof snRNPs andnon-snRNPprotein SC35was studiedwith theuse of immunofluorescent and immunoelectron microscopy[35] Following the activation of embryonic transcription inlate 2-cell mouse embryos splicing factors are revealed bothin IGCs and in association with the PFs

The Sm antigen of snRNPs and 227-trimethyl guanosine(TMG) cap characteristic for the mature snRNAs were local-ized in the nuclei of 1ndash4-cell porcine embryos at the ultra-structural level [43] Surprisingly unlike in bovine embryos(see below) immunoelectron microscopy also revealed theoccurrence of snRNPs in the NPBs of porcine embryosIn bovine early preimplantation embryos snRNPs werelocalized at different stages of ZGA [40] Before the onsetof transcription up to the 4-cell stage a diffuse labeling ofthe nucleoplasm was revealed After the beginning of tran-scription all 8-cell embryo nuclei weremarkedly stained andmRNPs were shown to concentrate at the periphery of chro-matin aggregates in association with the PFs Interestinglyin vitro-produced 8-cell embryos showed a higher degreeof chromatin condensation and a peripheral distribution ofchromatin blocks as compared with the embryos producedin vivo In 2- and 4-cell embryos an intensive anti-snRNPlabeling also characterized IGCs (nuclear speckles) whichwere often observed in the vicinity of theNPBs or even joinedto them at the 4-cell stage Using antibodies specific for theSm epitope of snRNPs the labeling was detected neitherin the NPBs during embryonic nucleologenesis nor in theresulting nucleoli

Analogous studies have been carried out later on bovineand caprine 2-cell embryos [41 61] The authors describedseveral types of extrachromosomal nuclear bodies (NBs)02ndash20120583m in diameter The most striking feature of theseNBs was the presence of proteins involved in pre-mRNAsplicing Some NBs having a rather dense finely fibrillarcomposition and thus named the dense bodies (DBs) were

shown to contain the Sm-antigen Moreover more numerousNBs differed morphologically from the former by a muchlooser composition of fibrillogranular elements (called loosebodies LBs) This type of the NBs in addition to the Sm-antigen contained the non-snRNP splicing factor SC35 amarker of IGCsspeckles Both types of the NBs were distin-guished clearly from the NPBs both morphologically and bythe absence of NPB immunolabeling with antibodies againstpre-mRNA splicing factors Apart from the NBs describedabove a high concentration of snRNPs was revealed in rathersmall approximately 005 120583m in diameter morphologicallyand poorly defined domains named small snRNP-enrichedareas (SSA) These domains housed a set of nuclear proteinstypical for the CBs including the CB signature protein coilinHowever it is still elusive whether the LBs correspond to thecanonical IGCs of other cells In the same way it is unknownwhether the DBs or SSA resemble the CBs in all respects

With use of different approaches including light andelectron immunocytochemistry and in situ hybridization thedynamics of the CBs andor IGCs has been studied in theearly embryos of the mouse [24 34 62] and the goldenhamster [44] In the mouse the CBs are already present in1-cell embryos before major transcription activation On thecontrary in hamster embryos they appear during the 2-cellstage after the onset of transcription On the other handhamster 1-cell embryos already display prominent IGCswhereas typical IGCs appear only at the 2-cell stage in themouse Thus the assembly of both CBs and IGCs seemsto be independent of the onset of transcriptional activityin the early mammalian embryo [44] Zatsepina et al [62]have shown in early mouse embryos heterogeneity of CB-like(coilin-containing) nuclear bodies both in size andmolecularcomposition So anti-Sm antibody was shown to stain smallcoilin-positive foci present in 1-cell and early 2-cell embryosand the periphery of significantly larger bodies present inmiddle and late 2-cell embryos


Little is known about mammalian embryo nuclear struc-tures that virtually could correspond to perichromatin gran-ules (PGs) It is suggested that the structure of PGs arises dueto the packaging of PFs and the PGs participate in transientstorage of mRNPs (in the form of heterogeneous nuclear(hn) RNPs) andor also in mRNP nucleoplasmic transport[63] Typical mammalian somatic PGs contain some splicingsnRNAs [64] and snRNP proteins [65]

Numerous small PG-like granules approximately 150 nmin diameter resembling similar structures in the nucleus ofantral oocytes [66] have been described in the nuclei ofmouse 2-cell embryos [9] Since the 4-cell stage the numberof the PGs decreases in the nuclei of mouse blastomeresand becomes comparable with their number in somatic cellsTaking into account the gradual increasing of embryonicRNA synthesis at this period the authors believe that at leasta part of the PGs in 2-cell mouse embryos contain maternalhnRNPs that will migrate into the cytoplasm or degrade Itcannot be excluded that some RNAs present in the PGs ofmouse embryos may play a regulatory role

The nature of larger RNP-containing granules 30ndash50 nmin diameter that have been described in the nuclei of 2ndash8-cellmouse embryos [9] remains unknown

4 Some Proteins of the Exon-ExonJunction Complex

In our group we also studied nuclear distribution of somerepresentative proteins constituting the exon-exon junctioncomplex (EJC) [45 46 67]TheEJC is amultiprotein complexthat is loaded onto mRNA during splicing at a preciseposition upstream of exon-exon junctions [68] Amongstother functions the EJC enhances nuclear export of mRNAproviding a platform to bind export factors [69] Core EJCproteins including Y14 [70] provide stable association of theEJC with mRNA whereas EJC shell proteins (eg AlyREF)serve as adaptors providing a link between splicing andexport [71]

In the nuclei of mouse embryos at the different stagesof ZGA the EJC core protein Y14 the shell protein Alyand the essential mRNA export factor NXF1TAP weredetected in transcriptionally inert 1-cell embryos but theintensity of fluorescence increased in late 2-cell embryosthat are transcriptionally active [67] Possible association ofNXF1TAPwith nuclear speckles was also studied at the 2-cellstage [45] In transcriptionally active embryos NXF1TAPwas detected in the vicinity of the IGCsspeckles ratherthan inside these nuclear domains Artificial inhibition oftranscription by drugs resulted in significant accumulation ofNXF1 in enlarged speckles

We also studied possible spatial interactions betweenthe EJC proteins and nuclear actin with the use of Forsterresonance energy transfer (FRET) [46] Two patterns of theFRET signal were detected in transcriptionally active nucleiof 2-cell embryos for the pairs actin-Y14 actin-AlyREFand actin-NXF1TAP FRET areas were revealed both ran-domly distributed in the nucleoplasm and in association

with NPBs The means of FRET efficiency exceeded 25ndash30 in separate areas We supposed that FRET signalsin the nucleoplasm correspond to transcriptionally activechromatin zones The presence of Y14 and NXF1TAP inthe NPBs was confirmed at the ultrastructural level [46]however it is hard to explain detection of FRET in thesenuclear structures of the embryos The FRET pattern typicalfor transcriptionally active embryos was being retainedafter artificial suppression of transcription by drugs Inthe specimens digested by RNAse FRET efficiency wasdecreased significantly showing the spatial interactionsbetween EJC proteins and actin occur in an RNA-dependentmanner

5 Closing Remarks

Thenucleus of earlymammalian embryos is a highly dynamicsystem The bulk of studies were devoted to the dynamicsof intranuclear accumulation of the revealed antigens butnot to their association with nuclear domains Availabledata on the domain organization of mammalian embryonuclei are not comprehensive except for nucleolus thegenesis of the CBs and IGCsspeckles was described in themouse and the golden hamster As concerns the embryosof farm animals real relationships between multifariousnuclear bodies knownunder various names and the canonicalnuclear domains of somatic cell have to be ascertainedHigh rates of nuclear structure reorganization together withdifferent chronologies that characterize early embryogenesisin various organisms make a comparative analysis of embryonuclear domains difficult Further studies in this field wouldbe helpful to form an integral concept of the structuraland functional compartmentalization of the nucleus as wellas to extend our knowledge on the mechanisms of earlyembryogenesis in mammals

Conflict of Interests

The authors declare no conflict of interests in this study Noauthor of this paper has a direct financial relation with thecommercial identitiesmentioned in the paper thatmight leadto a conflict of interests

Acknowledgment

Thisworkwas supported by the granting program ldquoMolecularand Cell Biologyrdquo of Russian Academy of Sciences

References

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[2] H Brahms J Raymackers A Union F de Keyser L Meheusand R Luhrmann ldquoThe C-terminal RG dipeptide repeats ofthe spliceosomal Sm proteins D1 and D3 contain symmetricaldimethylarginines which form a major B-cell epitope for anti-Sm autoantibodiesrdquo The Journal of Biological Chemistry vol275 no 22 pp 17122ndash17129 2000


[3] D L Spector X-D Fu and T Maniatis ldquoAssociations betweendistinct pre-mRNA splicing components and the cell nucleusrdquoThe EMBO Journal vol 10 no 11 pp 3467ndash3481 1991

[4] D Bogolyubov I Stepanova and V Parfenov ldquoUniversalnuclear domains of somatic and germ cells some lessons fromoocyte interchromatin granule cluster and Cajal body structureand molecular compositionrdquo BioEssays vol 31 no 4 pp 400ndash409 2009

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[6] L L Hall K P Smith M Byron and J B Lawrence ldquoMolecularanatomy of a specklerdquo Anatomical Record A Discoveries inMolecular Cellular and Evolutionary Biology vol 288 no 7 pp664ndash675 2006

[7] J G Gall ldquoCajal bodies the first 100 yearsrdquo Annual Review ofCell and Developmental Biology vol 16 pp 273ndash300 2000

[8] Z Nizami S Deryusheva and J G Gall ldquoThe Cajal body andhistone locus bodyrdquo Cold Spring Harbor Perspectives in Biologyvol 2 no 7 Article ID a000653 2010

[9] S Fakan and N Odartchenko ldquoUltrastructural organization ofthe cell nucleus in earlymouse embryosrdquo Biology of the Cell vol37 no 3 pp 211ndash218 1980

[10] C Bouniol E Nguyen and P Debey ldquoEndogenous tran-scription occurs at the 1-cell stage in the mouse embryordquoExperimental Cell Research vol 218 no 1 pp 57ndash62 1995

[11] F Aoki D M Worrad and R M Schultz ldquoRegulation oftranscriptional activity during the first and second cell cyclesin the preimplantation mouse embryordquo Developmental Biologyvol 181 no 2 pp 296ndash307 1997

[12] N Minami T Suzuki and S Tsukamoto ldquoZygotic gene activa-tion andmaternal factors inmammalsrdquo Journal of Reproductionand Development vol 53 no 4 pp 707ndash715 2007

[13] W Tadros and H D Lipshitz ldquoThe maternal-to-zygotic transi-tion a play in two actsrdquoDevelopment vol 136 no 18 pp 3033ndash3042 2009

[14] L Li P Zheng and J Dean ldquoMaternal control of early mousedevelopmentrdquo Development vol 137 no 6 pp 859ndash870 2010

[15] T Hamatani M G Carter A A Sharov and M S H KoldquoDynamics of global gene expression changes during mousepreimplantation developmentrdquo Developmental Cell vol 6 no1 pp 117ndash131 2004

[16] F Zeng and R M Schultz ldquoRNA transcript profiling duringzygotic gene activation in the preimplantation mouse embryordquoDevelopmental Biology vol 283 no 1 pp 40ndash57 2005

[17] O Oslashstrup G Olbricht E Oslashstrup P Hyttel P Collas andR Cabot ldquoRNA profiles of porcine embryos during genomeactivation reveal complex metabolic switch sensitive to in vitroconditionsrdquo PLoS ONE vol 8 no 4 Article ID e61547 2013

[18] E M Tompson E Legouy and J-P Renard ldquoMouse embryosdo not wait for the MBT chromatin and RNA Polymeraseremodeling in genome activation at the onset of developmentrdquoDevelopmental Genetics vol 22 no 1 pp 31ndash42 1998

[19] O Oslashstrup I S Andersen and P Collas ldquoChromatin-linkeddeterminants of zygotic genome activationrdquoCellular andMolec-ular Life Sciences vol 70 no 8 pp 1425ndash1437 2013

[20] J-E Flechon and V Kopecny ldquoThe nature of the ldquonucleolusprecursor bodyrdquo in early preimplantation embryos a review offine-structure cytochemical immunocytochemical and autora-diographic data related to nucleolar functionrdquo Zygote vol 6 no2 pp 183ndash191 1998

[21] P Hyttel ldquoNucleolus formation in pre-implantation cattle andswine embryosrdquo Italian Journal of Anatomy and Embryologyvol 106 supplement 2 no 2 pp 109ndash117 2001

[22] J Laurincik and P Maddox-Hyttel ldquoNudeolar remodeling innuclear transfer embryosrdquo Advances in Experimental Medicineand Biology vol 591 pp 84ndash92 2007

[23] S Bellier S Chastant P Adenot M Vincent J P RenardandO Bensaude ldquoNuclear translocation and carboxyl-terminaldomain phosphorylation of RNA polymerase II delineate thetwo phases of zygotic gene activation in mammalian embryosrdquoThe EMBO Journal vol 16 no 20 pp 6250ndash6262 1997

[24] I Bogolyubova and D Bogolyubov ldquoAn immunocytochemi-cal study of interchromatin granule clusters in early mouseembryosrdquo BioMed Research International vol 2013 Article ID931564 8 pages 2013

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[26] I O Bogolyubova ldquoTranscriptional activity of nuclei in 2-cellblockedmouse embryosrdquoTissue and Cell vol 43 no 4 pp 262ndash265 2011

[27] D Vautier D Besombes D Chassoux F Aubry and P DebeyldquoRedistribution of nuclear antigens linked to cell proliferationand RNA processing in mouse oocytes and early embryosrdquoMolecular Reproduction andDevelopment vol 38 no 2 pp 119ndash130 1994

[28] D Vautier P Chesne C Cunha A Calado J-P Renard andMCarmo-Fonseca ldquoTranscription-dependent nucleocytoplasmicdistribution of hnRNP A1 protein in early mouse embryosrdquoJournal of Cell Science vol 114 no 8 pp 1521ndash1531 2001

[29] D M Worrad P T Ram and R M Schultz ldquoRegulation ofgene expression in the mouse oocyte and early preimplantationembryo developmental changes in Sp1 and TATA box-bindingprotein TBPrdquoDevelopment vol 120 no 8 pp 2347ndash2357 1994

[30] K Wang F Sun and H Z Sheng ldquoRegulated expression ofTAF1 in 1-cell mouse embryosrdquo Zygote vol 14 no 3 pp 209ndash215 2006

[31] E Gazdag A RajkovicM E Torres-Padilla and L Tora ldquoAnal-ysis of TATA-binding protein 2 (TBP2) and TBP expressionsuggests different roles for the two proteins in regulation of geneexpression during oogenesis and early mouse developmentrdquoReproduction vol 134 no 1 pp 51ndash62 2007

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[34] J Ferreira andM Carmo-Fonseca ldquoThe biogenesis of the coiledbody during early mouse developmentrdquo Development vol 121no 2 pp 601ndash612 1995

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[36] I O Bogolyubova N A Bogoliubova D S Bogolyubov andV N Parfenov ldquoNuclear structure in early mouse embryosa comparative ultrastructural and immunocytochemical studywith special emphasis on the lsquo2-cell block in vitrorsquordquo Tissue andCell vol 38 no 6 pp 389ndash398 2006


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[38] M Biggiogera T E Martin J Gordon F Amalric and SFakan ldquoPhysiologically inactive nucleoli contain nucleoplas-mic ribonucleoproteins immunoelectronmicroscopy ofmousespermatids and early embryosrdquo Experimental Cell Research vol213 no 1 pp 55ndash63 1994

[39] A J Watson K E Wiemer M Arcellana-Panlilio and G ASchultz ldquoU2 small nuclear RNA localization and expressionduring bovine preimplantation developmentrdquoMolecular Repro-duction and Development vol 31 no 4 pp 231ndash240 1992

[40] V Kopecny S Fakan A Pavlok et al ldquoImmunoelectronmicroscopic localization of small nuclear ribonucleoproteinsduring bovine early embryogenesisrdquo Molecular Reproductionand Development vol 29 no 3 pp 209ndash219 1991

[41] V Kopecny M Biggiogera J Pivko et al ldquoThe cell nucleusin early bovine and caprine preimplantation embryos finestructural cytochemistry and immunoelectron microscopyrdquoEuropean Journal of Cell Biology vol 70 no 4 pp 361ndash372 1996

[42] R S Prather and L F Rickords ldquoDevelopmental regulation ofan snRNP core protein epitope during pig embryogenesis andafter nuclear transfer for cloningrdquo Molecular Reproduction andDevelopment vol 33 no 2 pp 119ndash123 1992

[43] V Kopecny M Biggiogera J Laurincik et al ldquoFine structuralcytochemical and immunocytochemical analysis of nucleicacids and ribonucleoprotein distribution in nuclei of pigoocytes and early preimplantation embryosrdquo Chromosoma vol104 no 8 pp 561ndash574 1996

[44] J Ferreira and M Carmo-Fonseca ldquoNuclear morphogenesisand the onset of transcriptional activity in early hamsterembryosrdquo Chromosoma vol 105 no 1 pp 1ndash11 1996

[45] I Bogolyubova D Bogolyubov and V Parfenov ldquoLocalizationof poly(A)+ RNA and mRNA export factors in interchromatingranule clusters of two-cell mouse embryosrdquo Cell and TissueResearch vol 338 no 2 pp 271ndash281 2009

[46] I Bogolyubova G Stein and D Bogolyubov ldquoFRET analysisof interactions between actin and exon-exon-junction complexproteins in early mouse embryosrdquo Cell and Tissue Research vol352 no 2 pp 277ndash285 2013

[47] J Greenblatt ldquoRNA polymerase II holoenzyme and transcrip-tional regulationrdquo Current Opinion in Cell Biology vol 9 no 3pp 310ndash319 1997

[48] V E Myer and R A Young ldquoRNA polymerase II holoenzymesand subcomplexesrdquoThe Journal of Biological Chemistry vol 273no 43 pp 27757ndash27760 1998

[49] T I L Lee and R A Young ldquoTranscription of eukaryoticprotein-coding genesrdquo Annual Review of Genetics vol 34 pp77ndash137 2000

[50] A S Neish S F Anderson B P Schlegel W Wei andJ D Parvin ldquoFactors associated with the mammalian RNApolymerase II holoenzymerdquo Nucleic Acids Research vol 26 no3 pp 847ndash853 1998

[51] C Rachez and L P Freedman ldquoMediator complexes andtranscriptionrdquo Current Opinion in Cell Biology vol 13 no 3 pp274ndash280 2001

[52] M E Dahmus ldquoReversible phosphorylation of the C-terminaldomain of RNA polymerase IIrdquo The Journal of BiologicalChemistry vol 271 no 32 pp 19009ndash19012 1996

[53] S Buratowski ldquoProgression through the RNA Polymerase IICTD CyclerdquoMolecular Cell vol 36 no 4 pp 541ndash546 2009

[54] M Heidemann C Hintermair K Voszlig and D Eick ldquoDynamicphosphorylation patterns of RNA polymerase II CTD duringtranscriptionrdquo Biochimica et Biophysica Acta vol 1829 no 1 pp55ndash62 2013

[55] S Fakan ldquoPerichromatin fibrils are in situ forms of nascenttranscriptsrdquo Trends in Cell Biology vol 4 no 3 pp 86ndash90 1994

[56] M J Goddard andH PM Pratt ldquoControl of events during earlycleavage of the mouse embryo an analysis of the lsquo2-cell blockrsquordquoJournal of Embryology and Experimental Morphology vol 73pp 111ndash133 1983

[57] J J Qiu W W Zhang Z L Wu Y H Wang M Qian and Y PLi ldquoDelay of ZGA initiation occurred in 2-cell blocked mouseembryosrdquo Cell research vol 13 no 3 pp 179ndash185 2003

[58] D B Bregman L Du S van der Zee and S L WarrenldquoTranscription-dependent redistribution of the large subunit ofRNA polymerase II to discrete nuclear domainsrdquo Journal of CellBiology vol 129 no 2 pp 287ndash298 1995

[59] M J Mortillaro B J Blencowe X Wei et al ldquoA hyperphos-phorylated form of the large subunit of RNA polymerase IIis associated with splicing complexes and the nuclear matrixrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 93 no 16 pp 8253ndash8257 1996

[60] N Saitoh C S Spahr S D Patterson P Bubulya A F Neuwaldand D L Spector ldquoProteomic analysis of interchromatin gran-ule clustersrdquo Molecular Biology of the Cell vol 15 no 8 pp3876ndash3890 2004

[61] V Kopecny M Biggiogera J Pivko et al ldquoFine-structural cyto-chemical and immunocytochemical observations on nuclearbodies in the bovine 2-cell embryordquo Zygote vol 8 no 4 pp315ndash328 2000

[62] O Zatsepina C Baly M Chebrout and P Debey ldquoThe step-wise assembly of a functional nucleolus in preimplantationmouse embryos involves the cajal (coiled) bodyrdquoDevelopmentalBiology vol 253 no 1 pp 66ndash83 2003

[63] Y Daskal ldquoPerichromatin granulesrdquo inThe Cell Nucleus vol 8pp 117ndash137 Academic Press New York NY USA 1981

[64] N Visa F Puvion-Dutilleul J-P Bachellerie and E PuvionldquoIntranuclear distribution of U1 and U2 snRNAs visualizedby high resolution in situ hybridization revelation of a novelcompartment containing U1 but not U2 snRNA in HeLa cellsrdquoEuropean Journal of Cell Biology vol 60 no 2 pp 308ndash321 1993

[65] E Puvion A Viron C Assens E H Leduc and P JeanteurldquoImmunocytochemical identification of nuclear structures con-taining snRNPs in isolated rat liver cellsrdquo Journal of Ultrastruc-ture Research vol 87 no 2 pp 180ndash189 1984

[66] F Palombi and A Viron ldquoNuclear cytochemistry of mouseoogenesis 1 Changes in extranucleolar ribonucleoprotein com-ponents through meiotic prophagerdquo Journal of UltrastructureResearch vol 61 no 1 pp 10ndash20 1977

[67] I O Bogolyubova and D S Bogolyubov ldquoLocalization ofmRNA export factors in early mouse embryosrdquo in HOAJBiology vol 1 no 11 2012

[68] H le Hir E Izaurralde L E Maquat and M J MooreldquoThe spliceosome deposits multiple proteins 20ndash24 nucleotidesupstream of mRNA exon-exon junctionsrdquo The EMBO Journalvol 19 no 24 pp 6860ndash6869 2000

[69] H le Hir D Gatfield E Izaurralde andM JMoore ldquoThe exon-exon junction complex provides a binding platform for factorsinvolved in mRNA export and nonsense-mediated mRNAdecayrdquoThe EMBO Journal vol 20 no 17 pp 4987ndash4997 2001


[70] T Oslash Tange T Shibuya M S Jurica and M J MooreldquoBiochemical analysis of the EJC reveals two new factors anda stable tetrameric protein corerdquo RNA vol 11 no 12 pp 1869ndash1883 2005

[71] Z Zhou M-J Luo K Straesser J Katahira E Hurt and RReed ldquoThe protein Aly links pre-messenger-RNA splicing tonuclear export in metazoansrdquo Nature vol 407 no 6802 pp401ndash405 2000

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

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Zoology


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Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


experimental models in order to analyze nuclear distributionof gene expression factors that virtually may associate withthe universal andor specific nuclear domains ZGA is one ofthe key points of the maternal-to-zygotic transition (MZT)that involves a set of structural molecular and biochemicalrearrangements [10ndash14] However the majority of studiesconcerned first of all the changes of gene expression patterns[15ndash17] as well as the processes of chromatin remodelingduring realization of ZGA events (for reviews see [18 19])The relationships between these processes and the distribu-tion of the essential components of nuclear metabolism weredescribed with a lesser extent

Here we review available literary data on nuclear distribu-tion of some essential molecular components related to RNApolymerase II-dependent transcription and mRNA process-ing at different stages of ZGA in mammals The dynamicsof the nucleolar components in mammalian embryos wasstudied better [20ndash22] and remains beyond the scope of thepresent review

2 RNA Polymerase II and BasalTranscription Factors

The association of RNA polymerase II (RNAP II) with manyfactors to form a polyfunctional holoenzyme is requiredfor transcription [47 48] Besides core RNAP II enzymethis complex includes basal transcription factors mediatorcomplexes and transcription coactivators [49ndash51] The largeRNAP II subunit (RPB1) contains the carboxy-terminaldomain (CTD) consisting of multiple heptapeptide repeatswith the consensus sequence YSPTSPS Dynamic phospho-rylation of the CTD determines the progression of the RNAPII transcription cycle from initiation through elongation totermination [52ndash54]

Posttranslational modifications of RNAP II holoenzymewere studied in mouse and rabbit preimplantation embryoswith the use of western blot analysis and immunofluores-cent microscopy [23] In this study a set of the followingmonoclonal antibodies was applied 8WG16 revealing theunphosphorylated CTD CC-3 directed against a phospho-rylated epitope of the CTD and Pol33 against an internalepitope distinct from the CTD thus recognizing two forms ofRPB1 simultaneously The authors have eventually suggestedthat phosphorylation of the CTD might control ZGA inthe embryos of mammals with different ZGA chronologiesSeveral hours after fertilization the CTD was found dephos-phorylated Dephosphorylation of the CTD occurs before theonset of a period characterized by a weak transcriptionalactivity (minor ZGA) Then the CTD lacks immunologicaland drug-sensitivity characteristics related to its phospho-rylation status and RNAP II gradually translocates intothe nuclei At the major ZGA phase corresponding the2-cell stage in mouse and the 8ndash16-cell stage in rabbitphosphorylation pattern of the CTD was close to thatobserved in somatic cells The authors also reported thatactinomycin D does not prevent a new phosphorylationpattern of RNAP II at the onset of the major ZGA Asa result a phosphorylated embryogenesis-specific RNAP II

isoform was identified This RNAP II isoform is insensitiveto DRB a CTD-kinase inhibitor since it lacks a phosphoepi-tope generated by TFIIH-associated kinase phosphorylationFinally the authors have concluded that nuclear translocationof RNAP II and CTD phosphorylation might be majordeterminants of ZGA The possible association of differentRNAP II isoforms with intranuclear structures of mouse andrabbit preimplantation embryos was not explored in detail inthis study

In our immunocytochemical experiments we used anaffinity purified polyclonal serum to reveal the hyperphos-phorylated form of RNAP II in mouse embryos This formof RNAP II was detected before the major ZGA phasenamely in early 2-cell embryos [24] While ZGA proceedsRNAP II was found in association with perichromatin fibrils(PFs) [25] which are referred to as the ultrastructural ldquoinsitu formsrdquo of nascent pre-mRNA transcripts [55] Besidesthe hyperphosphorylated RNAP II was detected in nuclearspeckles identified by the presence of the SR protein SC35The intensity of anti-RNAP II immunostaining in speckleswas being increased towards the end of ZGA [24] (Figure 1)If ZGA is delayed for example in embryos under the so-called ldquo2-cell block in vitrordquo (for details see [56 57]) thehyperphosphorylated form of RNAP II begins to accumulatein enlarged nuclear speckles [26] (Figure 2)

Worrad et al [29] have explored the concentration of thetranscription factors Sp1 and TBP (TATA-binding subunit ofTFIID) in 1-cell mouse embryos The authors showed thatconcentration of both factors drastically increases in a time-dependent fashion after fertilization during the 1-cell stageSix hours following the formation of pronuclei this increasecontinued and by the G2 phase the pronuclear concentrationof Sp1 and TBP was very similar to that observed in 2-cellembryos In addition concentration of both transcriptionfactors was greater in the male pronucleus and the differencewas more pronounced for TBP

Later TBP and TBP-associated factor 1 (TAF1) wererevealed in mouse zygotes [30] TAF1 was not detectedjust after fertilization in transcriptionally silent cells butit is expressed in pronuclei reaching the maximum beforethe onset of ZGA TAF1 and TBP shared similar dynamicsof expression patterns In 4 h after fertilization anti-TAF1immunofluorescent signal was not registered The signalbecame obvious in a portion of embryos in 6 h after fertil-ization in the majority of embryos in 9 h and in all embryosin 11 h TBP was revealed in some embryos for the first timein 4 h after fertilization and in almost all pronuclei 1 h laterIn male pronuclei transcriptional activity can be registeredearlier Respectively both factors initially appear in malerather than in female pronuclei The authors have supposedthat the deficiency of transcription machinery might be areason for the limitation of transcription at the beginning ofembryogenesis

In addition to TBP Gazdag et al [31] have revealedthe related protein TBP2 also called TRF3 (TBP-relatedfactor 3) in mouse 1-2-cell embryos They also reportedthat TBP is expressed in the oocytes at the beginning offolliculogenesis but cannot be detected during further stagesof oocyte development and becomes abundant again only


32h SC35 Merge

(a) (a998400) (a998400998400)

(b) (b998400) (b998400998400)

46h

PolII


46 in vitro SC35

(a)

PolII

(b)

72 in vitro

(c) (d)








TBP IF 1-cell [28]

Sp1 TBP

IFMicroinjection of


1-cell-2-cell [29]


Splicing factors

Mouse



U1 snRNAU1 snRNP










ISHIF




[34][28]











(a) (a998400) (a998400998400)

(b) (b998400) (b998400998400)

24h SC35 Merge

46h

TFIID
















5 Closing Remarks




Acknowledgment


References


















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


32h SC35 Merge

(a) (a998400) (a998400998400)

(b) (b998400) (b998400998400)

46h

PolII


46 in vitro SC35

(a)

PolII

(b)

72 in vitro

(c) (d)








TBP IF 1-cell [28]

Sp1 TBP

IFMicroinjection of


1-cell-2-cell [29]


Splicing factors

Mouse



U1 snRNAU1 snRNP










ISHIF




[34][28]











(a) (a998400) (a998400998400)

(b) (b998400) (b998400998400)

24h SC35 Merge

46h

TFIID
















5 Closing Remarks




Acknowledgment


References


















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







TBP IF 1-cell [28]

Sp1 TBP

IFMicroinjection of


1-cell-2-cell [29]


Splicing factors

Mouse



U1 snRNAU1 snRNP










ISHIF




[34][28]











(a) (a998400) (a998400998400)

(b) (b998400) (b998400998400)

24h SC35 Merge

46h

TFIID
















5 Closing Remarks




Acknowledgment


References


















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (a998400) (a998400998400)

(b) (b998400) (b998400998400)

24h SC35 Merge

46h

TFIID
















5 Closing Remarks




Acknowledgment


References


















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










5 Closing Remarks




Acknowledgment


References


















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Review Article Nuclear Distribution of RNA Polymerase II and...

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Transcript of Review Article Nuclear Distribution of RNA Polymerase II and...