BMSC 320 Lecture 22: mRNA Processing PDF
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Uploaded by CrisperGyrolite5945
2024
Dr. Anderson
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Summary
This document is lecture notes on mRNA processing, covering topics like modifications, processing events, and stability in both prokaryotes and eukaryotes. The lecture discusses the different stages of mRNA processing, from capping and splicing to termination and poly-A tailing, and how these processes affect mRNA stability and translation efficiency.
Full Transcript
Nov. 4, 2024 MSC 320: Nucleic Acids, From Central B Dogma to Human Disease WF 9:30 - 10:20 M Lecturer: Dr. Anderson Lecture 22: mRNA Processing part 1 mRNA processing - A series of extensive modifications In prokaryotic mRNA, transcription and translation occurs simultaneous...
Nov. 4, 2024 MSC 320: Nucleic Acids, From Central B Dogma to Human Disease WF 9:30 - 10:20 M Lecturer: Dr. Anderson Lecture 22: mRNA Processing part 1 mRNA processing - A series of extensive modifications In prokaryotic mRNA, transcription and translation occurs simultaneously in the nucleoid, so their mRNA are transcribed in a “ready to read” state and do not need further processing. In eukaryotes, further processing of the mRNA is needed to create a mature transcript. ○ They are transcribed as a pre-mRNA in the nucleus. ○ Modifications occur at the 5’ and 3’ ends ○ Splicing of exons together to remove the introns ○ Export from nucleus and localization in the cytoplasm ○ Ensuring translation of messages you want and inhibiting translation of messages you don’t want (ex. We may want a delayed transcription). ○ Control of turnover - stability or degradation. Overview of RNA Processing A mature mRNA contains: ○ A 5’ cap and 5’ untranslated region (UTR) = stabilizes the transcript and makes its translations efficient. ○ Start codon and open reading frame (ORF) termination at a stop codon. ○ A 3’ UTR (location for binding different factors related to gene regulation) and poly-A tail (not encoded for by DNA) = stabilizes the transcript and makes its translations efficient. Order of processing events: 1. Capping 2. Splicing (during transcription) 3. Termination/Polyadenylation 4. Splicing (after termination) mRNA (and other RNA) is inherently unstable T he structure of the RNA, specifically its ribose sugar (extra -OH), makes it more prone to breakage, unstable and reactive. Ribonucleases in cells can act on the 5’ end, 3’ end (exonucleases), or at internal sequences or structures (endonucleases). They degrade RNA to recycle them overtime unlike DNA. There are certain drugs or heat shock mutations that are used to terminate transcription either immediately or slowly. ○ Instantaneous (broken lines) lead to levels of existing pre-mRNA to quickly drop as they are being processed into mature RNA. Overtime, the mature RNA levels also gradually decrease because they are being degraded. ○ Slow inhibition (solid lines) lead to a more tapered decrease in the level of pre-MRNA and mature RNA. Increasing RNA stability or protecting it from degradation means one transcript can yield more rounds of translated proteins. mRNA is unstable, but much more stable in Eukaryotes - Comparing the mRNA half-life in different organisms. E -coli mRNAs are degraded around 3-5 mins. S.cerevisiae mRNAs are degraded around 20-30 mins. ○ The tall bar on the >60 just represents all of the data over 60 mins. H. sapiens mRNA are degraded around 10+hours. 5’-end pre-mRNA Capping W e need to mask the 5’ end of the mRNA so they are not recognized as such. The 5’ end of a pre-mRNA is atriphosphateas the first ribonucleotide was ATP/CTP/GTP/CTP When a new transcript is ~20-30 bps long, capping enzymes are recruited to place a “cap” on the 5’ end to prevent its degradation by 5’ → 3’ exonucleases. ○ Capping doesn’t occur immediately, instead they wait until the transcript is beyond the abortive state, so they wait for around 20-30 bps. Multiple Steps involved: 1. First addition: Capping enzymes associated with the Pol II’s CTD when its ser5 are phosphorylated.Guanylyl transferasemakes a 5’ to 5’ bond between theinverted GTPand the 5’ end of the growing mRNA. a. There is no 5’ end on this transcript because of the addition of an inverted GTP, it has a 3’ end instead. 2. Second addition: The inverted-guanine is methylated at position 7 by guanine-7-methyl transferase. a. Recall that RNA is unstable because of that extra -OH in its ribose, by methylating this, we make the RNA more stable. 3. Third addition: methyl transferase can methylate the 2’ -OH of the fist two 2’ end nucleotides. (Only in multicellular organisms) Cap Complexity Varies by Organism Complexity Yeast only methylated the position 7 of the GTP. (7-m-G-cap) Most plants and animals also methylate the 2’ carbon of deoxyribose of the +1 nucleotide (cap–1) Vertebrates also methylate the 2 carbon of the +2 nucleotide (cap-2), an RNA in a vertebrate without this methylation is assumed to be a viral RNA. These methylations also help our mRNA to be recognized as our own; viral RNA do not have these methylations in their RNA triggering the innate interferon response. Cap-Binding Complex The capping occurs in the nucleus so the virus cannot use the proteins needed because they are not present in the cytoplasm where the viral RNA are. O nce the 5’ end of the mRNA is processed it can be distinguished from the transcripts from Pol I or Pol III. Nuclear CBC (cap-binding complex) binds, mediating the association of different proteins for their different pathways ○ The cap is needed to recruit the spliceosome. ○ The cap is needed for pre-mRNA 3’ end processing to begin. ○ The cap stabilizes mRNA ○ It allows for RNA export from the nucleus to the cytoplasm. ○ Pioneer round of translation. ○ Nonsense mediated decay ○ mRNA biogenesis Termination and The 3’ Poly–A-Tail R ecall, termination in prokaryotic transcription by intrinsic structures (ex. Rho- independent) or protein-mediated (ex. Rho-dependent) mechanism that “pulls” the mRNA from RNApol. In eukaryotes, transcription terminates when the mRNA is cleaved from the transcript. The pol II continues to transcribe until theXrn2 5’ → 3’ exonucleasetravels along the remaining RNA (like a lit fuse) degrading it until it reaches the pol II making the pol II to dissociate -Torpedo model There is a poly(A) signal and a Poly (A) site in the DNA sequence. ○ Poly (A) signal recruits proteins needed for tailing. ○ Poly(A) site marks where the adenylation will start. Termination - Detection The Pol II CTD has associated proteins,CstF and CPSF, involved in transcription termination and polyadenylation of the 3’ end of the mRNA. They are facilitated by the lack of Ser-5-P and the presence of Ser-2-P. ○ CstF = cleavage stimulation factor ○ CPSF = cleavage and polyadenylation specificity factor. ○ The termination being dependent on the state of the pol II CTD prevents early termination if a mutation occurs where the poly (A) site or the poly (A) signal is moved closer to the promoter. Once RNA pol II transcribes a signal in the pre-mRNA, these factors will move from the CTD of the pol II to the pre-mRNA. ○ CPSF binds to theAAUAAA (poly-A signal) ○ CstF binds to a downstreamG/U rich region. (poly-A-signal) Four more factors bind: ○ CF I (cleavage factor I) ○ CF II (cleavage factor II) ○ PAP (poly-A polymerase) - do not need a template to add adenine. As long as they have a 3’ end to bind to, they can just add Adenine. ○ PAB (poly-A- binding protein) CF I and II cut the pre-mRNA between the 2 boxed sequences. Termination - poly-A-Tailing The mRNA is cleaved ~35 bases past the AAUAAA. Poly-A polymerase (PAP) adds a few A’s, PAB needs to bind, if not the PAP loses its activity. Poly-A binding protein (PAB) binds this short run of A’s to stimulate further polyadenylation. PAP activity is enhanced when both CPSF and PABP are present and quickly adds up to 250 more adenines. (in a non-template manner) At the 3’ end, the tail can shorten without affecting any important sequences (until it gets too short) so a longer tail can help the RNA to persist. mRNA needs Caps and Tails m RNA half-life is extended when caps and tails are added. Modified transcripts are translated faster than unmodified transcripts. An experiment was conducted to show the difference in the rate of translation between an mRNA that had a cap and tail, just a cap, just a tail and with no cap nor tail. ○ Cap and tail = very bright ○ Just cap = lowers by a factor of 10, meaning that there’s a lower grade of translation ○ Just tail = even lower, shows that translation is cap-dependent because it is required to recruit ribosomes needed. ○ No cap or tail = very very low. ○ They also looked at the half-life of the different mRNA, results showed the mRNA without cap or tail to have the lowest half-life. Cap and Tail Translational Synergy Once the mature mRNA is exported out of the nucleus, the cap and tail stabilize and enhance its translation by formingclosed-loops. ○ 5’cap is bound byeukaryotic intrinsic factors (eIF)that associate with the 3’ poly-A tail via PABP. ○ Initial protein synthesis rate becomes faster as ribosomes are recycled. Destabilizing this system with excess caps or tails blocks the recycling.
