RNA Processing Lecture 2021 PDF

**[Eurkaryotic RNA Polymerases]** - Pol I transcribes rRNA (precursors to 18S and 28S) - Pol II transcribes mRNAs, miRNAs, snRNAs, snoRNAs, lincRNA - Pol III transcribes tRNAs, 5S rRNA, other small RNAs, ~~gRNA~~ - All of these RNAs have to be processed post-transcriptionally in vari...

**[Eurkaryotic RNA Polymerases]** - Pol I transcribes rRNA (precursors to 18S and 28S) - Pol II transcribes mRNAs, miRNAs, snRNAs, snoRNAs, lincRNA - Pol III transcribes tRNAs, 5S rRNA, other small RNAs, ~~gRNA~~ - All of these RNAs have to be processed post-transcriptionally in various ways - ***Important concept***: Post-transcriptional processing of RNA is not encoded in the genome! ie pseudouridine (Ψ), the polyA tail, etc. **[Ribosomal RNA (rRNA) Processing]** - In prokaryotes: 16S, 23S, 5S (tRNAs are found interspersed) - [In eukaryotes: 18S, 5.8S, 25S] - This is the one exception to the monocistronic rule for eukaryotic genes; rRNA is polycistronic (one transcript encodes multiple genes). Important for controlling dosage, if making 18S rRNA you need 25S rRNA as well---always have the same amounts. - 5S rRNA (transcribed by Pol-III and is least processed!) - 1% of genome and repeated in all genomes (*E. coli* has 7 copies, eukaryotes have hundreds) - RNA Pol I has a very strong promoter, 40% of transcription is devoted to making the ribosome - Co-transcriptional association with dynamic processing factors and ribosomal proteins (RPs) - Modification: base pairing interactions with snoRNAs to guide enzymes - in the **nucleolus** by proteins and snoRNAs (**snoRNPs**), which cleave rRNAs and modify bases: - H/ACA → pseudouridine (looks like U but with a C-ribose connection instead of N-ribose) - Box C/D → ribose-2-O-methylation - RNAse III-family enzyme cleaves hairpins leaving a 2nt 3'OH overhang **[Transfer RNA (tRNA) Processing]** - Most heavily processed RNA - Anti-codon loop must be precise - T loop defined by a T residue instead of a U 1. RNaseP (a ribozyme) removes 5' leader sequence 2. RNAseZ removes 3' end UU 3. [CCA-adding enzyme] adds CCA to 3' end 4. TSEN complex excises introns 5. Chemically modify nucleotides including adenosine deaminase - Modification: mainly on global and local structure, the modifying enzymes use structure and sequence for specificity Diagram Description automatically generated - Generally, [nucleotide modifications] have diverse consequences (Met of 2'OH and pseudoUridylation are most common): - Modulation of base-paring and decoding e.g. Wobble pairing - Structure stabilization - Binding of specific readers - 2'OMe provides RNA stability **[Messenger RNA (mRNA) Processing]** - Consists of capping, splicing and polyadenylation. - Regulates nuclear export, stability, expression of desired protein, loading by the ribosome and degradation - mRNAs have short half-life (hours) and high turnover; compared to rRNA which is stable and can last days - mRNA comprises about 3% of total cell RNA - **pre-mRNA capping -- 7-methyl guanosine cap** - Remove 5'P, add GMP 5'-5', methylate G - Occurs co-transcriptionally (as soon as RNA 5' end of mRNA emerges, the cap is put on) - Functions: - Translation initiation (recognition of 7MeG by cap complex) - Stabilizes mRNA (5'-5' bond is stable, can't be seen by 5' =\> 3' exonucleases) - Directs splicing and maturation (defines first exon) - Directs export from the nucleus - **Polyadenylation** - PolyA tail **is not encoded in DNA**. Signal for addition is [AAUAAA]...CA followed by U/GU-rich region, binds CPSF - Co-transcriptionally: - Cleavage factors cut after CA (CFI, CFII, CPSF, CstF finds AAUAAA) - **PolyA Polymerase(PAP)** slowly adds polyA tail - **PABP** binds tail; help make it resistant to nucleases - Functions: - Regulates termination of transcription - 3' intron removal - mRNA export - Alternative polyA sites can produce proteins with different C-terminuses (eg. secreted vs membrane-bound Ig) - Stabilize mRNA (protect from 3' exonuclease) - Translation initiation (PABP binds cap complex) - Use polyA to purify (with polyU/T columns or using polyT primers in RT-PCR) - [Exception! Histone mRNA doesn't have polyA tail] - Short life, regulated by cell cycle - Have special 3' end that helps connect them to cell cycle machinery - 3' end generated by snRNA reaction (U7 snRNA), forms a stem loop - **SLBP** (Stem-loop binding protein) binds to protect instead of PABP - - - - - - - - - - - - 1. Branch site **A** 2'OH attacks 5' **GU** located just outside exon 1 2. 3'OH of 5' end attacks 5' end of exon 2 3. Products are spliced exons and lariat  - 1. 2. 3. 4. 5. - - - - - - - - - - - - - **[RNA editing]** - - - - - - - - - m6A is abundant - may represent spill over activity from tRNA modifying enzymes - Multi-modification can also be used to mark self to prevent immune activation **[RNA decay]** - Post-transcriptionally regulated process that is critical to steady-state RNA levels and regulation - Also needed for RNA quality control and removal of foreign RNA - mRNA lives differ significantly. Can be regulated by: - De-adenylation and de-capping (rate-limiting by endonuclease) then exonucleolytic activity (canonical RNA decay) - siRNA or miRNA - Nonsense Mediated Decay (NMD), No-go deay (NGD), Non-stop decay (NSD)... \[covered in another lecture\] - Bacterial mRNA decay: RNase E cleavage - Euk mRNA decay: De-adenylation and de-capping (rate-limiting by endonuclease, regulated by 3'UTR structure and sequence) then exonucleolytic activity (canonical RNA decay) **[The Complexity of Euk expression: they likely evolved as part of defense mechanism against genomic parasites! ]**