BIOL 200 Lecture PL1 - RNA Processing 2024

 REGISTER AS A TEAM OR INDIVIDUALLY WIN A NIGHT AT THE HYATT + MORE PRIZES! SIGN UP USING OUR @GLOBALBRIGADESMCGILL REGISTRATION FORM BELOW!  CKUT hasn’t received a fee increa...

 REGISTER AS A TEAM OR INDIVIDUALLY WIN A NIGHT AT THE HYATT + MORE PRIZES! SIGN UP USING OUR @GLOBALBRIGADESMCGILL REGISTRATION FORM BELOW!  CKUT hasn’t received a fee increase since 2012. We need your vote in the SSMU referendum! Scan to vote! Follow @saveckut on instagram and visit ckut.ca/yes BIOL 200 Lecture PL1 8 November 2024 RNA processing--part I Processing of precursor mRNAs involves four distinct events Lodish, Fig. 9-2 (1) Capping of the 5’ end of the pre-mRNA  5’ capping of pre-mRNA is coupled to transcription Precursor mRNAs (pre-mRNA) are modified at their 5’ end after the first ~25 nucleotides are transcribed when transcription pauses. A 7’ methylguanylate cap is added to the 5’ terminal nucleotide through an unusual 5’- 5’ triphosphate linkage. In animal cells and in higher plants the 2’ hydroxyl of the ribose group of the first base is methylated. In vertebrates the second nucleotide is also methylated. Addition of the cap: -protects the pre-mRNA from degradation -facilitates nuclear export -assists recognition by translation factors Lodish, Fig. 5-26  How is RNA capping linked to transcription? The large subunit of RNA Pol II has an extended carboxy- terminal domain (CTD) composed of Ser-rich 7-mer repeats YSPTSPS52 in humans YSPTSPS~26 in yeast CTD Ser5 is phosphorylated during the pausing stage of transcription CDK7 CDK9 Cole and Cowling (2008) 9: 810-815 CTD P-Ser5 stimulates capping Capping enzymes bind to phosphorylated Ser5 and are activated. This ensures that only RNAs transcribed by Pol II (mostly mRNAs) are capped, because Pol I and Pol III don’t have CTDs. RNA processing and transcription elongation are tightly coupled through the CTD Serine 2 phosphorylation recruits additional proteins: Splicing factors Polyadenylation factors Export factors Capping Enzyme CDK7 CDK9 Cole and Cowling (2008) The CTD can interact with many different proteins because it forms a large (65-130 nm) extended structure Lodish, Fig. 9-4 Processing of precursor mRNAs involves four distinct events Lodish, Fig. 9-2 (2) Cleavage and (3) polyadenylation Cleavage and polyadenylation are tightly coupled RNA polymerase II is released from the DNA template at regions called the terminator. The terminator is some distance downstream from what will be the 3’ end of the mRNA. A 3‘ AAUAAA xxxxx G/U sequence is recognized by cleavage and polyadenylation factors (CPSF, CStF, CFI, CFII). This sequence is called the poly(A) signal. The 3’ end of the pre-mRNA is cleaved at the poly(A) site and then poly(A) polymerase (PAP) catalyzes the formation of a poly (A) tail. Lodish, Fig. 9-15 (top half)  Cleavage and polyadenylation are tightly coupled Polyadenylation occurs in two phases: A slow phase mediated by poly(A) polymerase (PAP) during which approximately 12 A residues are added on to the cleaved 3’ end. This structure is recognized by nuclear poly (A) binding protein (PABPN1) which catalyzes the rapid addition of ~200 A residues. Lodish, Fig. 9-15 (bottom half) Are all mRNA transcripts polyadenylated? All mRNAs are polyadenylated except histone mRNAs…they have unique secondary structure in their 3’ UTRs -Other mRNAs that lack a poly(A) tail are rapidly degraded within the nucleus. -Although histone mRNAs lack a poly(A) tail, they are not degraded. Pre-mRNAs associate with nuclear proteins and form particles These particles include hnRNPs (heterogeneous nuclear ribonucleoprotein particles). hnRNP proteins contribute to further steps of RNA processing (polyadenylation, export to cytoplasm, splicing). hnRNP proteins are modular, containing one or more RNA binding domains and often also one or more intrinsically disordered protein domains.  RNA binding proteins have characteristic domains Other common RNA binding domains: KH domains RGG Domains Pumilio/PUF Domains Lodish, Fig. 9-6 Three functions of hnRNPs Association with hnRNPs prevent the formation of sequence-specific secondary structures through base pairing. The hnRNPs impose a uniform structure that processing enzymes can recognize. hnRNPs can regulate pre-mRNA splicing. Many pre- mRNAs can be spliced in more than one way, and hnRNPs bound in or near splice sites can promote or repress their use. hnRNPs function in RNA transport as some can cycle in and out of the nucleus. Processing of precursor mRNAs involves four distinct events Lodish, Fig. 9-2 (4) RNA splicing From pre-mRNA to mRNA: splicing Unlike bacterial genes, most eukaryotic genes have introns. Exons are the regions of a pre-mRNA that are present in the mature mRNA, whereas introns get spliced out from the pre-mRNA and are not part of the mature mRNA. Lodish, older edition, see also Fig. 8-18 -DNA segments that encode introns are not junk- they can encode regulatory information. -Introns are more common and generally larger in higher eukaryotes.  Early evidence for splicing: DNA is not fully contiguous with mRNA Introns were discovered because a discrepancy was observed between mRNA size and gene size. They were also visualized by hybridization experiments. mRNA of the adenovirus hexon gene hybridized to the DNA fragment containing the hexon gene forms an RNA- DNA hybrid. Intron sequences in the DNA loop out. Lodish, Fig. 9-7 Intron borders are highly conserved Comparison of a large number of introns indicated that their border sequences are conserved. These features are useful (but not sufficient) for the in silico prediction of mRNA sequences based on genomic sequencing data. Splice Donor Splice Acceptor Lodish, Fig. 9-7c The GU AG rule… The A of the Branch point is also highly conserved… Intron splicing involves two trans- esterification reactions The hydroxyl group of the residue at the branch point attacks the 5’ phosphate group of the first intron residue (G) leading to formation of a “lariat”. The free 3’ end of the preceding exon then attacks the 5’ phosphate group of the first residue in the following exon (second trans-esterification), resulting in the joining of the two exons and the release of the intron lariat. Lodish, Fig. 9-8 Small nuclear RNAs and splicing factor proteins are necessary for correctly targeting splicing events and bringing the ends of the intron together. Five small nuclear RNAs (snRNAs) and ~50 associated proteins participate in pre-mRNA splicing. U1 snRNA base-pairs with pre-mRNA around the 5’ splice site and U2 snRNA) base-pairs with pre-mRNA around the branch point. Lodish, Fig. 9-9a the branch point is not paired and bulges out!