1. Messenger RNA Processing II: Capping and Polyadenylation Chapter 15

2. Capping By 1974 - mRNA from a variety of eukaryotic species and viruses were found to be methylated - clustered at the 5’- end of mRNA - formed a structure called cap Cap Structure - The cap is made of 7- methylguanosine - m 7 G –Linkage is a triphosphate –Charge on the cap area is near -5

3. Reovirus Cap Structure The m 7 G contributes a positive charge Triphosphate linkage contributes 3 negative charges Phosphodiester bond contributes 1 negative charge Terminal phosphate contributes 2 negative charges

4. Cap Synthesis First step –RNA triphosphatase removes terminal phosphate from pre- mRNA –Then, guanylyl transferase adds capping GMP from GTP

5. Cap Synthesis Next, 2 methyl transferases methylate N 7 of capping guanosine and 2’-O-methyl group of penultimate nucleotide This occurs early in transcription, before chain is 30 nt long

6. Functions of Cap Caps serve at least four functions: –Protect mRNAs from degradation –Enhance translatability of mRNAs –Transport of mRNAs out of nucleus –Efficiency of splicing mRNAs

7. Polyadenylation Most eukaryotic mRNAs and their precursors have a chain of AMP residues about 250 nt long at their 3’-ends- called poly (A) -The process of adding poly(A) to RNA is called polyadenylation -Poly(A) is added posttranscriptionally by poly(A) polymerase Heterogeneous nuclear mRNA is a precursor to mRNA Function - Poly(A) enhances both the lifetime and translatability of mRNA

8. Basic Mechanism of Polyadenylation Transcription of eukaryotic genes extends beyond the polyadenylation site The transcript is: –Cleaved –Polyadenylated at 3’-end created by cleavage

9. Polyadenylation Signals An efficient mammalian polyadenylation signal consists of: –AAUAAA motif about 20 nt upstream of a polyadenylation site in a pre-mRNA –Followed 23 or 24 bp later by GU-rich motif –Followed immediately by a U-rich motif Variations on this theme occur in nature –Results in variation in efficiency of polyadenylation –Plant polyadenylation signals usually contain AAUAAA motif –More variation exists in plant than in animal motif –Yeast polyadenylation signals are even more different

10. Cleavage of Pre-mRNA Polyadenylation involves both: –Pre-mRNA cleavage –Polyadenylation at the cleavage site Cleavage in mammals requires several proteins –CPSF – cleavage and polyadenylation specificity factor –CstF – cleavage stimulation factor –CF I –CF II –Poly (A) polymerase –RNA polymerase II

11. Initiation of Polyadenylation Optimal signal for initiation of such polyadenylation of a cleaved substrate is AAUAAA followed by at least 8 nt When poly(A) reaches about 10 nt in length - further polyadenylation becomes independent of AAUAAA signal and depends on the poly(A) itself 2 proteins participate in the initiation process –Poly(A)polymerase –CPSF binds to the AAUAAA motif

12. Elongation of Poly(A) Elongation of poly(A) in mammals requires a specificity factor called poly(A)-binding protein II (PAB II) This protein –Binds to a preinitiated oligo(A) –Aids poly(A) polymerase in elongating poly(A) to 250 nt or more PAB II acts independently of AAUAAA motif –Depends only on poly(A) –Activity enhanced by CPSF

13. Polyadenylation Model Factors assemble on the pre-mRNA guided by motifs Cleavage occurs Polymerase initiates poly(A) synthesis PAB II allows rapid extension of the oligo(A) to full-length

14. Poly(A) Polymerase Structures of the enzymes predicted from the longest sequence includes: –RNA-binding domain –Polymerase module –2 nuclear localization signals –Ser/Thr-rich region – this is dispensable for activity in vitro

15. Turnover of Poly(A) Poly(A) turns over in the cytoplasm RNases tear it down Cytoplasmic Poly(A) polymerase builds it back up When poly(A) is gone mRNA is slated for destruction

16. Cytoplasmic Polyadenylation Maturation-specific polyadenylation of Xenopus maternal mRNAs in the cytoplasm depends on 2 sequence motifs: –AAUAAA motif near the end of mRNA –Upstream motif called the cytoplasmic polyadenylation element (CPE) UUUUUAU Or closely related sequence

17. Coordination of mRNA Processing Events Capping, polyadenylation and splicing - these processes are related Cap can be essential for splicing - but only for splicing the first intron Poly(A) can also be essential - but only for splicing out the last intron

18. mRNA-Processing Occurs During Transcription All three of the mRNA-processing events take place during transcription –Splicing begins when transcription is still underway –Capping When nascent mRNA is about 30 nt long When 5’-end of RNA first emerges from polymerase –Polyadenylation occurs when the still-growing mRNA is cut at the polyadenylation site

19. Binding of CTD of Rpb1 to mRNA-Processing Proteins The CTD of Rpb1 subunit of RNA polymerase II is involved in all three types of processing Capping, polyadenylating, and splicing enzymes bind directly to the CTD which serves as a platform for all three activities

20. Mechanism of Termination Termination of transcription by RNA polymerase II occurs in 2 steps: –Transcript experiences a cotranscriptional cleavage (CoTC) within termination region downstream of the polyadenylation site This occurs before cleavage and polyadenylation at the poly(A) site It is independent of that process –Cleavage and polyadenylation occur at the poly(A) site Signals polymerase to dissociate from template

21. Termination Signal CoTC element downstream of the polyadenylation site in the human b-globin mRNA is a ribozyme that cleaves itself –This generates a free RNA 5’-end –This cleavage is required for normal transcription termination –It provides an entry site for Xrn2, a 5’  3’ exonuclease that loads onto the RNA and chases RNA polymerase by degrading the RNA

22. Xrn2 - Exonuclease Xrn2 terminates transcription like a “torpedo” There is a similar torpedo mechanism in yeast where cleavage at poly(A) site provides entry for the 5’  3’ exonuclease Rat1 Rat1 degrades the RNA until it catches the polymerase and terminates transcription

23. Torpedo Model for Transcription Termination

24. Polyadenylation Polyadenylation is required for efficient transport of mRNAs from their point of origin in the nucleus to the cytoplasm

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