Formation of mRNA by RNAPII is linked with the recruitment of essential
processing factors, located at the place of RNA synthesis in the nucleus (Zhao et al. 1999, Glover-Cutter et al. 2008). Co-transcriptional recognition of regulatory factors involved in the process of cleavage and polyadenylation of nascent pre-mRNA, is mediated through interactions with RNAPII during transcription elongation. This provides a mechanistic explanation for the c end mRNA processing. The recruitment of polyadenylation factors to RNAPII and the type of promoter plays an important role in determining the efficiency of polyadenylation (Mapendano et al. 2010, sWang et al. 2010).
Eukaryotic DNA-dependent RNA polymerase (RNAPII) is initially recruited in a hypo- phosphorylated state to the promoter. It is composed of up to 12 polypeptides in which Rpb1 is the largest subunit and contains the catalytic activity (Hsin and Manley 2012). Nuclear polyadenylation is restricted to nascent transcripts of Pol II, by the interaction of cleavage and polyadenylation factors with the C terminal domain (CTD), a unique feature of RNAPII (Zhao et al. 1999). The carboxyl terminal domain (CTD) of the largest subunit Rbp1 of RNAP II is composed of conserved 52 heptapeptide repeats (N-Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7-C) in humans (Proudfoot and O'Sullivan 2002, Ryan et al. 2004, Glover-Cutter et al. 2008, Proudfoot 2011, Hsin and Manley 2012, Mayer et al. 2012) and each repeat consist of three serine
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residues reversibly phosphorylated throughout the process of transcription (Moore and Proudfoot 2009). The CTD determines the efficiency of all processing steps required for the formation of mature RNA, and its function is modulated by the different states of phosphorylation with the aid of kinases and phosphatases (Zhao et al. 1999, Hsin and Manley 2012). The CTD interacts with cleavage and polyadenylation factors and recruits them to RNAPII, especially if it is Ser 2 phosphorylated. In this way, RNAPII functions as part of the polyadenylation machinery (Yonaha and Proudfoot 2000, Proudfoot 2011, Hsin and Manley 2012). The phosphorylation on serine 5 (Ser5P) by TFIIH associated kinase Cdk7 (cyclin dependent kinase-7) is involved in early stages of elongation (Moore and Proudfoot 2009). Phosphorylation on serine 2 (Ser2P) by Cdk9 (Cyclin-Dependent kinase-9) or Cdk12 (Davidson et al. 2014) during late stages of transcriptional elongation recruits the cleavage and polyadenylation complex to the poly (A) signal through CTD- interacting domains (CIDs) mediating 3 formation of mRNAs, while polyadenosine binding protein (PABP) binds to the newly formed poly-A sequence (Shim et al. 2002, Glover-Cutter et al. 2008, Mapendano et al. 2010, Mayer et al. 2012). Ser5 is dephosphorylated by phosphatases during transcripton elongation, forming a pattern of phosphorylation in which Ser5-P is high at the start of transcription, while Ser2-P emerges at later stages of transcription.
The kinase activity of CDK9 (P-TEFb) and Cyclin T plays a regulatory role in transcriptional control of the elongating polymerase (Garriga and Grana 2004, Moore and Proudfoot 2009). A component of CFII, Pcf11 in mammals, has a CID (CTD interacting domain) and interacts with the Ser-2 phosphorylated CTD. This leads to recruitment of the polyadenylation complex, including CPSF1 and PCF11, to RNAP II as shown in figure 1.4 (Kuehner et al. 2011, Nagaike and Manley 2011). The phosphorylation of TFIIB serine residue 65 is important in the recruitment of CPSF and CstF to the promoter, just after the Ser5P phosphorylation of CTD RNAPII and before the initiation of transcription (sWang et al. 2010).
The last stages of co-transcriptional processing of an mRNA are important for preventing the formation of abnormal transcripts (Moore and Proudfoot 2009). For
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efficient transcription, termination by RNAPII cleavage of an mRNA is a necessary event (Moore and Proudfoot 2009, Proudfoot 2011). According to the torpedo model, endonucleolytic cleavage of pre-mRNA at the poly (A) signal during transcription is an essential step required for exonucleases activity of Xrn2 (Rat1 in yeast). Xrn2 polyadenylation- associated cleavage and contacts RNAP II, resulting in conformational changes and leading to the dissociation of nascent RNA from Pol II. Thus it leads to arrest of the elongation complex of Pol II and favours termination (Rosonina et al. 2006, Proudfoot 2011). The ability of polyadenylation factors such as Pcf11 to bind to the CTD is indeed a prerequisite for transcription termination (Hsin and Manley 2012).
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Figure 1.4: Mechanism of transcriptional coupled polyadenylation:
Polyadenylation factor CPSF is initially recruited to the promoter by TFIIB, a part of PIC, which is later transferred to the transcription complex of CTD (Ser5P) RNAP II at elongation initiation stage. As elongation proceeds, CstF is transferred to CTD RNAP II. As soon as the RNAP II transcribes the poly (A) signal, cleavage and polyadenylation factors are recruited on newly transcribed mRNA from the CTD (Ser2P) of RNAPII. Thus upon recognition of the poly(A) signal, transcriptional elongation pauses just next to the poly(A) signal, resulting in termination of transcription, leading to the cleavage of RNAPII associated 3’end of an mRNA. This in turn leads to the addition of poly (A) tail of ~200-250 residues by PAP bound by PABP. Assembly of poly (A) complex leads to the cleavage of an mRNA, which expels the CTD results in dissociation of pause Pol II from the template.
Abbreviations: AAUAAA (poly (A) signal), CstF (cleavage stimulating factor), CPSF
(cleavage/polyadenylation specificity factor), Pol II (RNA polymerase II), PAP (poly (A)- polymerase), PABP (Poly (A)-binding protein). TFIIB (Transcription factor IIB), CTD (C terminal domain).
Two possible mechanisms of transcriptional termination coupled to cleavage and polyadenylation have been reported. In most genes, a transcriptional pause site is located just next to the poly(A) signal at which cleavage occurs, while Pol II is still involved in transcription. (Zhao et al. 1999). Another class of termination occurs in the absence of a pause site downstream to the poly(A) signal, causing RNAPII to move further down the flanking region of an mRNA. An additional
termination sequence is found further downstream, which is known as the cotranscriptional cleavage (CoTC) sequence. The CoTC mediates a downstream cleavage event that precedes cleavage of the polyadenylation site. The degradation of nascent transcripts by Xrn2 at the P II
from DNA templates along pre-mRNAs, which are still linked to the CTD via poly(A) complexes (Moore and Proudfoot 2009, Proudfoot 2011).