(6.5.20)--RNAPolymeraseIIPhosphorylation.pdf

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1、Chapter 5 2012 Calvo and Garca,licensee InTech.This is an open access chapter distributed under the terms of the Creative Commons Attribution License(http:/creativecommons.org/licenses/by/3.0),which permits unrestricted use,distribution,and reproduction in any medium,provided the original work is pr

2、operly cited.RNA Polymerase II Phosphorylation and Gene Expression Regulation Olga Calvo and Alicia Garca Additional information is available at the end of the chapter http:/dx.doi.org/10.5772/48490 1.Introduction RNA polymerases(RNAPs)are among the most important cellular enzymes.They are present i

3、n all living organisms from Bacteria and Archaea to Eukarya and are responsible for DNA-dependent transcription.Although in Bacteria and Archaea there is only one RNAP,Eukarya possess up to three RNAPs in animals(I,II and III)and five in plants(IV and V)1-2.All of the RNAPs are evolutionarily relate

4、d and have common structural and functional properties.The minimally conserved structural organization is represented by the bacterial enzyme,which contains only 4 subunits(),whereas Archaea and Eukarya RNAPs are composed of 12 subunits(Rpb1-Rpb12)3.In prokaryotes,one RNAP transcribes all of the gen

5、es into all of the RNAs,however,in eukaryotes,this is achieved by three RNAPs.RNAPI transcribes genes that encode for 18S and 28S ribosomal RNAs;RNAPIII transcribes short genes,such as tRNAs and 5S ribosomal RNA,and RNAPII transcribes all protein-coding genes and genes for small noncoding RNAs(e.g.,

6、small nuclear RNAs(snRNAs)that are involved in splicing).The largest catalytic subunits of all three eukaryotic polymerases share homology among themselves and with the largest subunit of bacterial polymerase 4.Solely the largest subunit of RNAPII(Rpb1)contains an unusual evolutionarily conserved ca

7、rboxy-terminal domain(CTD)5,which is subjected to numerous post-translational modifications of extraordinary importance in gene expression regulation 6-8.RNAPII transcription plays a central role in gene expression and is highly regulated at many steps,such as initiation,elongation and termination.F

8、urthermore,phosphorylation of the Rpb1 CTD is known to regulate all of the transcription steps and coordinate these steps with other nuclear events.Prior to mRNA biosynthesis,RNAPII proceeds through several steps,such as promoter recognition,preinitiation complex(PIC)assembly,open complex formation,

9、initiation and promoter escape.This sequence of events is initiated by the binding of gene-specific activators and coactivators,which results in the recruitment of basal transcription machinery(i.e.,general transcription factors(GTFs):Protein Phosphorylation in Human Health 152 TFIIA,TFIIB,TFIID,TFI

10、IE,TFIIF,and TFIIH)and RNAPII to promoters 9-11.Basal transcription factors position RNAPII on promoters to form the PIC but also function at later steps,such as promoter melting and initiation site selection.Thereafter,initiation proceeds,and RNAPII leaves the promoter during promoter clearance and

11、 proceeds into processive transcript elongation.Finally,when the gene has been fully transcribed,transcriptional termination occurs,and RNAPII is released and recycled to reinitiate a new round of transcription 12-14.During its passage across a gene,RNAPII must overcome challenges.Initially,the poly

12、merase needs to escape from the promoter,and the synthesis of the pre-mRNAs must be tightly coupled to its subsequent processing(i.e.,capping,splicing,and polyadenylation).Then,initiation factors must be exchanged for elongation factors 15,which are thought to increase the transcription rate and RNA

13、PII processivity.In fact,recently,there has been an extraordinary increase in the number of proteins known to influence transcription elongation by avoiding transcriptional arrest,facilitating chromatin passage and mRNA processing 16-21,allowing mRNA packaging into a mature ribonucleoprotein(mRNP)an

14、d controlling mRNP quality and mRNA export 13,22-28.Therefore,the discovery of all of these factors has provided further evidence that the elongation phase is also highly regulated in eukaryotic cells and strictly coordinated with other nuclear processes 12-14.2.RNAPII CTD phosphorylation:The CTD co

15、de During the last two decades,gene expression studies have provided further evidence that many steps in gene expression,originally considered distinct and independent,are,in fact,highly coordinated,linked and regulated in a complex web of connections 29-30.The central coordinator that directs this

16、regulatory network(i.e.,from transcription initiation to termination and with pre-mRNA processing)in combination with many other nuclear functions is RNAPII,and the carboxy-terminal domain(CTD)of its largest subunit is of remarkable importance.CTD phosphorylation regulates and coordinates the entire

17、 transcription cycle with pre-mRNA processing,mRNA transport and with chromatin remodeling and modification 13.The CTD,therefore,has a critical integrating role in essentially all of the mRNA biogenesis steps,thus,it is subject to a dynamic regulation during the transcription cycle(i.e.,21,31-32).Th

18、erefore,RNAPII phosphorylation is one of the key processes in the regulation of transcription specifically and gene expression in general;consequently,deciphering the mechanisms that underlie RNAPII phosphorylation regulation has become one of the most studied issues in the field of gene expression.

19、RNAPII is comprised of 12 subunits(Rpb1-12)that are structurally and functionally conserved from yeast to mammals 33-34.In 1985,the largest subunit of RNAPII,Rpb1,from mouse and Saccharomyces cerevisiae,was cloned 4,35,and its sequence revealed that it contained a highly conserved carboxy-terminal d

20、omain(CTD).This domain has been extensively studied since then and,although it is a simple repetition in tandem of the heptapeptide consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7(YSPTSPS);Figure 1),the CTD has an extremely complex functionality.The consensus sequence is present in RNA Polymer

21、ase II Phosphorylation and Gene Expression Regulation 153 animals,plants,yeast,and in many protists 5,36-37,and it has been hypothesized that the CTD structure has originated through amplifications of a repetitive DNA sequence and that the number of repeats appears directly correlated with genomic c

22、omplexity(Figure 1A;38).For example,mouse and human CTDs contain 52 repeats 35,39-40;the Drosophila CTD contains 45 repeats 41;25-27 repeats are found in the yeast CTD(Figure 1A;4);and 15 repeats are found in protozoan CTDs 5,38.Although the CTD is completely dispensable for in vitro transcription,i

23、t is required for efficient RNA processing 17,42.In fact,the CTD is essential for cell viability because its deletion is lethal in mice,Drosophila and yeast,and partial truncations or site-specific mutations cause specific growth defects 5,42.Figure 1.Human and Saccharomyces cerevisiae Rpb1-CTDs.Ori

24、ginal studies showed that two RNAPII forms can be differentiated in SDS-PAGE gels because of the different mobility of Rpb1 43.These two forms were termed RNAPIIA and RNAPIIO,and they differ in the extent of CTD phosphorylation.RNAPIIA is hypophosphorylated 44,and RNAPIIO is hyperphosphorylated 45.M

25、oreover,both forms,IIA and IIO,are functionally distinct because the IIA form is preferentially recruited to the promoter and associated with preinitiation complexes 46,whereas RNAPIIO functions during elongation,is highly phosphorylated 44 and thus requires de-phosphorylation to stimulate its recru

26、itment into the PIC complexes and to reinitiate a new round of transcription 47.We currently know that this earlier two-step transcription cycle Protein Phosphorylation in Human Health 154 model that is based on the two RNAPII forms is overly simple.Different phosphorylated forms of RNAPII are speci

27、fic and characteristic of the different steps that occur during the transcription cycle 48,and the correct progression of RNAPII through the transcription cycle is dependent on changes in the CTD phosphorylation status.Differential CTD phosphorylation promotes the exchange of initiation and elongati

28、on factors during promoter clearance 15,the exchange of elongation and 3-end processing factors at termination 49,as well as RNAPII recycling 50 and,moreover,links pre-mRNA processing and other nuclear events with transcription 17,42.Therefore,the CTD phosphorylation cycle is very complex.It is wide

29、ly known that the three serines(i.e.,Ser2,Ser5,and Ser7)7,51,the tyrosine 52-53 and the threonine 54 in each repeat can be phosphorylated.Additionally,both proline residues can be isomerized by a prolyl isomerase 55.Moreover,glycosylation of serines and threonine can also occur 8,and in human cells,

30、the CTD can be methylated at some of the degenerate repeat sites 56.The multitude of possible CTD modifications,especially Ser phosphorylations,in combination with the numerous repetitions,gives rise to a wide range of variations(i.e.,phosphorylation patterns)that have been termed the“CTD code”(Figu

31、re 2)6-8.Figure 2.The CTD Code.Only Ser(S)and Thr(T)phosphorylation sites have been considered.CTD glycosylation has not been considered 57 because this modification is mutually exclusive of phosphorylation 8.n:number of consensus repetitions.The RNAPII CTD code determines and coordinates the timely

32、 sequential recruitment of required specific factors during the transcription cycle.Therefore,the CTD functions as a scaffold that coordinates mRNA biogenesis,such as transcription initiation 58,promoter clearance 59,elongation 60,and termination 31,61-62,as well as RNA processing 17,21,30 and snRNA

33、 and snoRNA gene expression 63-65 by recruiting the appropriate set of factors when required during active transcription.These factors recognize CTD RNA Polymerase II Phosphorylation and Gene Expression Regulation 155 phosphorylation patterns either indirectly or directly by contacting phosphorylate

34、d residues.Among the CTD-associated factors are export and histone modifier factors and DNA repair factors 21.2.1.Ser2P and Ser5P,and to a lesser extent,Ser7P,are the main determinants of the CTD code To determine precisely which serine residues are phosphorylated in a particular repeat has been cha

35、llenging because of the numbers of phospho-acceptor amino acid residues and consensus motif repetitions(Figure 1).However,studies involving chromatin immunoprecipitation with specific monoclonal antibodies have provided evidence that differential phosphorylation of the Ser residues coincides with th

36、e temporal and spatial recruitment of different factors 8,32,48,66-67.In fact,these antibodies have been largely used to decipher and characterize the role of CTD phosphorylation during the transcription cycle and in gene expression regulation 8,32,68.Antibodies that selectively recognize either Ser

37、2 or Ser5 phosphorylation(i.e.,Ser2P or Ser5P,respectively)were the first residues to be described 66;phosphorylation of these two residues has been extensively studied,and they have been considered as the two main determinants of the CTD code 6.It is widely known that CTD phosphorylation switch fro

38、m Ser5 to Ser2 during the course of transcription and is subject to a dynamic regulation during the whole transcription cycle 69-71.The level of Ser5 phosphorylation peaks early in the transcription cycle and remains constant or decreases as RNAPII progresses to the 3 end of the gene (Figure 3);48,6

39、7,72).In contrast,Ser2 phosphorylation is the predominant modification in the coding and 3-end gene regions and occurs simultaneously with productive elongation 31,48,73.On the other hand,de-phosphorylation of Ser5 occurs during the initiation-elongation transition and throughout the entire elongati

40、on step,whereas Ser2 de-phosphorylation occurs at the end of transcription to recycle the polymerase and reinitiate a new round of transcription.Therefore,reversible phosphorylation/de-phosphorylation of the CTD plays a significant role in modulating the transcription cycle 31-32.Most recently,the u

41、se of new anti-CTD monoclonal antibodies has demonstrated that Ser7,which is the most degenerate position of the CTD 41,can be phosphorylated during the transcription of snRNA genes and protein-coding genes 64,68,74.Subsequently,this mark increases the complexity of the CTD code 7-8.Ser7 phosphoryla

42、tion is mediated by the same kinase 74-75,although,at least in Saccharomyces cerevisiae,Ser7P appears not to be dephosphorylated by the same Ser5 phosphatase(see below)76.The first study on Ser7 phosphorylation provided further evidence that this modification is functionally important for transcript

43、ion and processing of snRNAs 8,64 and hypothesized that the CTD code could be gene-transcription dependent.In mammals,Ser7P peaks at the promoter region of snRNA genes but is enhanced toward the 3 end of protein-coding genes 68.Recent genome-wide distribution studies in yeast have provided further e

44、vidence that Ser7P in protein coding genes occurs early during transcription initiation and is maintained during the entire transcription cycle.In fact,Ser7P is not only maintained,but it is also generated de novo during transcription elongation.Additionally,it has been hypothesized that Ser7 phosph

45、orylation could facilitate elongation and suppress cryptic transcription 77.Protein Phosphorylation in Human Health 156 Figure 3.RNAPII CTD phosphorylation profile in Sacharomyces cerevisiae.During transcription initiation and promoter escape RNAPII CTD is phosphorylated on Ser5(Ser5P)48,78.Concurre

46、ntly,Ser7 is phosphorylated(Ser7P),establishing a bivalent mark at both protein-coding and noncoding genes 74-76.Shortly after promoter dissociation,Ser5P is rapidly removed while phosphorylated Ser2(Ser2P)and Ser7P continue to accumulate 70,77.Finally,all CTD marks are rapidly removed at the end of

47、 transcription,and the hypophosphorylated RNAPII(in grey)is ready to assemble into the pre-initiation complex and re-initiate transcription 73,79-80.Small circles represent phosphorylated serine residues(green cirlces for Ser5P,blue circles for Ser7P and red circles for Ser2P).Differently colored bi

48、g circles represent the distinct phosphorylated forms of RNAPII during initiation,elongation and termination.TSS:transcription start site;p(A):polyadenylation site.2.2.Tyrosine 1 and Threonine 4 can also be phosphorylated Tyrosine 1(Tyr1)is evolutionarily conserved and present in all of the 52 repea

49、ts of the mammalian CTD,and in all of the 26-27 repeats of the yeast CTD.Although,it is well known that Tyr1 is susceptible to phosphorylation by tyrosine kinases in vivo 52-53 and that Tyr1 mutations are lethal in yeast 81,the function of this modification is unknown.Additionally,threonine 4(Thr4)i

50、s also subjected to phosphorylation,at least in mammalian and in yeast cells 54,82,and recently it has been demonstrated that phosphorylation of the Thr4 residues is required specifically for histone mRNA 3 end processing,which facilitates the recruitment of 3 processing factors to histone genes,and