The process of nuclear polyadenylation is a multiple step event that involves several protein complexes of multiple subunits (de Klerk et al., 2012, Ghoshal and Jacob, 1991). Proteomics experiments indicate the cleavage and polyadenylation complexes consist of more than 80 proteins in humans (Shi et al. 2009, Nunes et al. 2010). The main components of these multi protein complexes are highly conserved and comprise several protein complexes flanking the cleavage site, including CPSF, CstF, cleavage factors I and II (CFIm and CFIIm) and poly (A) polymerases (PAP), as
summarised in table 3. For the process of cleavage and polyadenylation, the assembly of these protein complexes in mammals is initiated by the interaction of CPSF and CstF to core sequences on premature mRNAs (Zhao et al. 1999, Takagaki and Manley 2000). There are some scaffolding proteins that lack RNA binding function and only serve as a binding site for other regulatory elements such as symplekin and CstF-3 (Tian and Manley 2013).
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The UGUAN UT‘
recruits an additional heterodimeric core factor, CFIm, which stimulates the process
of cleavage and polyadenylation in the absence of the conserved AAUAAA hexamer. The role of CFIIm is still unknown (Danckwardt et al. 2008, Nunes et al. 2010).
Table 3: List of factors involved in mammalian 3’ end cleavage and polyadenylation
Factors Subunits (KDa) HUGO Nomenclature Features CPSF (cleavage and polyadenylation specificity factor)
160 CPSF 1 Recognizes the poly (A) signal and interacts with CstF-77, PAP and CTD of RNAPII
100 CPSF 2 I ‘NA
73 CPSF 3
Endonuclease mediates the cleavage of pre-mRNA at 10-30 nucleotides downstream of the poly (A)
signal
30 CPSF 4 Binds to U rich elements on pre-mRNA
Fip1L - In vitro, FipL1 is responsible for recruitment of PAP thus link CPSF1 and PAP
CstF (cleavage stimulating factor) 50 CstF 1 M CTD
RNAPII thus facilitate assembly of other factors of polyadenylation machinery
64 CstF 2 Interact with G/U or U-rich downstream signals
77 CstF 3 Connect CstF-64 and CstF-50 and interacts with CPSF1
CFIm
(cleavage factor Im complex)
25 NUDT21
(CPSF5) Interact with upstream element (USE) containing U(G/A)UA, serves cru end processing
68 CPSF6 59 CPSF7 CFIIm (cleavage factor IIm complex) - PCF11 Unknown
Adapted from: (Colgan and Manley 1997, Zhang et al. 2005, Sagawa et al. 2011,
Charlesworth et al. 2013).
The most commonly found nucleotide followed next to cleavage site is cytosine (Tian and Manley 2013). Upon transcription at the poly (A) site by RNAPII CPSF, which was initially linked with the CTD, is recruited to poly (A) signal leading to RNA cleavage and termination of transcription (Glover-Cutter et al. 2008). Upon
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recognition, the recruitment of CPSF and CstF subunits to their respective sequence elements on pre-mRNA is assisted by CFIm, together with RNAPII, CF IIm and PAP, forming a competent cleavage complex as shown in figure 1.2. After the assembly of multi protein complexes to their specific cis acting regulatory sites on pre-mRNA, cleavage occurs which is followed by addition of the poly (A) tail (Zhao et al. 1999).
Figure 1.2: Schematic representation of factors involved in cleavage process:
Recruitment of protein factors of cleavage and polyadenylation machinery at their cognate sites on pre-mRNA for 3’ end processing. CPSF 1(160) specifically recognises poly (A) signal which associates with PAP and is involved in connecting CstF 3 (77) and PAP. CstF interact with GU rich region by CstF 2 (64). CF Im and CF IIm are also required for efficient cleavage process.
Abbreviations: AAUAAA (poly (A) signal), CstF (cleavage stimulating factor), CPSF
(cleavage/polyadenylation specificity factor), Pol II (RNA polymerase II), CTD (C-terminal domain), CF Im (cleavage factor I), CF IIm (cleavage factor II), PAP (poly(A)-polymerase).
Binding of PAP (poly (A)-polymerase) to RNA is weak and non-specific in the presence of Mn2+, however in combination with Mg2+ and CPSF, PAP becomes specific for AAUAAA and the complex of PAP, CPSF, CstF and RNA is stabilized,
C
A
CFII
mCFI
m3
1
AAUAAA
3
3
Cleavage site
1
4
2
Competent cleavage
complex
CstF
CPSF
F
ip
1
L
PAP
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mediating polyadenylation (Zhao and Manley 1996). Soon after, the endonucleolytic cleavage nuclear PAP adds 200-250~ adenosine residues as shown in figure 1.3 (Zarkower and Wickens 1987, Meijer et al. 2007, Cevher et al. 2010), which is bound by PABPN1 (Nuclear poly A binding protein). The first poly(A) polymerase (PAP alpha or PAPOLA) isolated from calf thymus, is thought to be the main mediator of nuclear polyadenylation by most mRNAs (Bardwell et al. 1990). It has been reported
that poly(A) polymerase is required for both cleavage and polyadenylation. A second canonical poly(A) polymerase, PAP gamma (PAPOLG) has also been linked
to nuclear polyadenylation (Ghoshal and Jacob 1991, Zhao and Manley 1996, Zhao et al. 1999).
Except for PABP, all protein factors are necessary for in vitro cleavage, but for in vitro polyadenylation only CPSF (Fip1L), PAP and PABP are required (Mandel et al. 2008). Nuclear poly(A) binding protein (PABPN1) controls the final length of the poly(A) tail, by disrupting the interaction of CPSF (Fip1L) and PAP, as the A 250 nucleotides by an unknown mechanism (de Klerk et al., 2012., Eckmann et al., 2011; Kuhn et al., 2009). The association of other proteins with polyadenylation machinery, such as nucleophosmin, plays a significant role in the regulation of length of poly (A) tail and in export of an mRNA from nucleus to cytoplasm (Sagawa et al. 2011). The binding of PABPN1 is weak and it is replaced by PABPC1 (cytoplasmic poly (A) binding protein) while still in the nucleus (Hosoda et al. 2006). After export, PABPC1 then interacts with eIF4G initiation complex bound to the cap of the mRNA, resulting in the formation of the competent closed loop complex and initiation of translation (Danckwardt et al. 2008).
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Figure 1.3: Process of nuclear cleavage and polyadenylation in eukaryotes:
Once the Trans-acting multiprotein complexes are assembled on their respective cis-acting RNA sequence elements, the pre-mRNA is cleaved endonucleolytically by CPSF-3 (CPSF73). This is followed by the addition of adenosine residues by PAP to form poly (A) tail, which is initially bound to PABPN1. Upon nuclear export PABPN1 is replaced by cytoplasmic PABPC. PABPC forms the closed loop ribonucleoprotein complex upon interaction with translation initiation factor eIFG, a part of the translaton initiation complex.
Abbreviations: USE (upstream sequence element), AAUAAA (poly(A) signal), DSE
(downstream sequence element), CstF (cleavage stimulating factor) (blue complex), CPSF (cleavage/polyadenylation specificity factor) (light blue complex), Pol II (RNA polymerase II), CF I (cleavage factor I) (green complex), CF II (cleavage factor II), PAP (poly(A)- polymerase), PABPN1 (nuclear poly(A)-binding protein), PABPC (cytoplasmic poly(A)- binding protein) 4A, 4E, 4G (translation initiation factors).
1.3.2 Regulation of gene expression by co-transcriptional processing of the