Tutorial PL2-3 2024 RNA Processing PDF

RNA Processing II & Nucleocytoplasmic Transport PL2/3 Holly J | BIOL200 | November 20th 2024  Housekeeping December 4th: No lecture End of term instructor review Questions must be submitted before December 3rd at noon Sessions will...

RNA Processing II & Nucleocytoplasmic Transport PL2/3 Holly J | BIOL200 | November 20th 2024  Housekeeping December 4th: No lecture End of term instructor review Questions must be submitted before December 3rd at noon Sessions will be recorded Back to regular tutorial/lecture schedule (tutorials are one week behind) RNA Processing II  mRNA Processing PL1 Pre-mRNA to mature mRNA Modifications at the 5’ and 3’ needs of the pre-mRNA are important for stability and protection 3 major co-transcriptional steps: 1. 5’ Capping 2. 3’ Cleavage and Polyadenylation 3. RNA Splicing  5’ Capping PL1 methylguanalate cap Nascent mRNA emerges from RNA exit channel (25 bps) 5’ cap is added by capping enzyme that interacts with RNA Pol II CTD Protects the mRNA, facilities nuclear export, and allows recognition by translation initiation factors  3’ Cleavage & Polyadenylation PL1 Poly(A) signal = AAUAAA, + G/U is recognized by cleavage and polyadenylation factors (CPSF, CStF, CFI, CFII) 1 1. 3 ‘ end is cleaved 2 3 2. 3’ end is recognized by Poly A Polymerase (PAP) 4 3. Slow Phase: 12 A residues added to 3’ end 4. Rapid Phase: PolyA Binding Protein (PABP) adds around 200 A residues  mRNA Splicing PL1/2 1. U1 (intron splice site) and U2 (branch point) interact with mRNA 2. Recruitment of U4, U5, U6 3. U1 & 4 exit (active splicesesome) 4. Transesterification Reactions 1. OH at branch point attacks 5’ phosphate at the 1st intron residue, forming the lariat 2. 3’ end of exon attacks 5’ end of following exon 5. Release of mature mRNA, snRNAs and intron  Self Splicing Introns In some cases, introns have the capacity self splice, without the need for external snRNAs (not a common mechanism)  mRNA Splicing Specificity The exon cross recognition complex U2AF: Helps with splicing efficiency SR Proteins: Contain RRM domains & ESEs: Exonic splicing enhancers, arginine/serine rich domains, mediate the sequences within the exons that cooperative binding of U1 to the 5’ splice site and promote splicing U2 to the branch point by binding to exonic splicing enhancers (ESEs)  Alternative Splicing The presence of multiple introns allows for the expression of multiple variations of a protein from a single gene -> protein isoforms  Alternative Splicing Ex: Sexual differentiation in Drosophila Sxl protein is only present in female embryos (early development) Binding site = intronic splicing silencer (blocks use of splice site) Later in development, sex-lethal gene is active in males and females  Alternative Splicing Ex: Sexual differentiation in Drosophila Males: In the absence of early Sxl protein, sex-lethal mRNA is spliced to make mRNA that contains a stop codon No functional Sxl protein -> Splicing of tra exon 1 to exon 2 - > no functional transformer protein  Alternative Splicing Ex: Sexual differentiation in Drosophila Females: In the presence of early Sxl protein, exon 3 of the sex-lethal mRNA is skipped Functional Sxl protein -> binding of Sxl to ISS skips exon 2 -> functional transformer protein  Alternative Splicing Ex: Sexual differentiation in Drosophila Dsx protein (double-sex gene) Females: complex of tra, Rbp1 and tra2 direct splicing, cleavage and alt polyadenylation at 3’ end of exon 4 = short, female specific Dsx Binding site = exotic splicing enhancer Males: no tra, so exon 4 is skipped = longer, male specific Dsx  RNA Editing Not a mutation! Sequence of the mature mRNA sometimes differs from the DNA’s coding regions sequence Adenosine to Inosine (A -> I) OR Cytosine to Uracil (C -> U) Ex: Editing of apoB pre-mRNA changes C->U at position 6666 in intestinal cells Shorter apoB-48 has N- terminal domains that associates with lipids Nucleo-Cytoplasmic Transport Nuclear Pore Complex Proteins are synthesized in the cytoplasm -> need a way to exit the nucleus, and potentially go back in NPC: made up of nucleoporins, making up a nuclear basket and aqueous pore FG nucleoporins: extended disordered regions (Phe-Gly repeats + hydrophilic regions) form permeability barrier Molecules up to 60kDA freely diffuse, anything bigger must be transported  Nuclear Import Cytoplasm -> Nucleus Nuclear Transport Receptor: Bind to the NLS domain to facilitate transport through the pore (FG domains) Proteins wanting to enter the nucleus require a nuclear localization signal (NLS) 1. Free importin binds to NLS on cargo protein, 5 forming complex 1 2. Complex interacts with FG repeats in NPC channel 2 3. Once in the nucleus, importin interacts with Ran- GTP causing the release of the cargo protein (decreased affinity) 4. Importin-Ran-GTP complex diffuses back to 4 cytoplasm 6 5. Ran interacts with Ran-GAP, hydrolyzes GTP to 3 GDP, importin is released 6. Ran-GDP goes back to nucleus, interacts with RAN: Monomeric G Protein Ran-GEF, causing GDP to be exchanged for GTP  Nuclear Export Nuclear Transport Receptor: Nucleus -> Cytoplasm Bind to the NLS domain to facilitate transport through the pore (FG domains) Proteins wanting to exit the nucleus require a nuclear export signal (NES) 1. Free exportin1 forms complex with Ran-GTP 3 and cargo protein in the nucleus (increased affinity) 2. Complex interacts with FG repeats in NPC 4 channel 2 3. Once in the cytoplasm, complex encounters Ran-GAP, stimulating hydrolysis of GTP, cargo is released 4. Exportin1 and Ran-GDP are transported back to the nucleus 5. Ran-GEF stimulates conversion of Ran-GDP- 1 5 >GTP  mRNP Export Ran-Independent Mechanism mRNP Exporter: Large NXFI (export factor) subunit, small NXT1 (export transporter) subunit Once processing of mRNA is completed in the nucleus, it remains associated with proteins in a messenger ribonucleoprotein (mRNP) complex Exporter cooperatively binds RNA that has specific mRNP proteins that associate with pre-mRNAs during elongation (before processing) Form a domain that interacts with FG repeats, allowing for diffusion across NPC Questions?