Post-Transcriptional Gene Control PDF

Post-Transcriptional Gene Control  RNA Processing & Post-Transcriptional Gene Control Most RNAs are processed in the nucleus from primary transcripts before export to cytoplasm Note: – Locations of various mechanisms – Types of RNA polymerases...

Post-Transcriptional Gene Control  RNA Processing & Post-Transcriptional Gene Control Most RNAs are processed in the nucleus from primary transcripts before export to cytoplasm Note: – Locations of various mechanisms – Types of RNA polymerases and RNA  Pre-mRNA Processing Eukaryotic premature mRNA transcript synthesized by RNA polymerase II is processed: 1. 5’ Capping 2. 3’ Polyadenylation 3. Intron Removal & Exon Splicing  Pre-mRNA & hnRNP pre-mRNA: nascent mRNA transcripts of protein-coding genes (mRNAs never exist as free RNA molecules but are always associated with proteins) - RNP: ribonucleoprotein complexes (pre-mRNA +nuclear proteins) – hnRNA: heterogeneous nuclear RNA (pre-mRNA & other nuclear RNA) hnRNP: heterogeneous ribonucleoprotein particles – combination of hnRNA + proteins – hnRNA is associated with an abundant set of nuclear proteins that contain conserved RNA-binding domains  hnRNP Functions of nuclear hnRNP: – prevent folding of pre-mRNA into secondary structures that may inhibit its interactions with other proteins pre-mRNA in hnRNP present a more uniform substrate for processing steps such as splicing – transport of mRNA out of the nucleus nuclear proteins remain associated until the mRNA is exported to the cytosol; mRNA then associates w/ cytoplasmic proteins!  5’ Capping shortly after RNA pol II initiates transcription, when pre-mRNA transcript is ~ 25-30 bases long, 7-methylguanylate cap added to 5’ end (5’-5’ linkage) and first couple ribonucleotides are methylated on 2’ OH of ribose Purpose: – protects 5’ end from enzymatic exonuclease degradation in nucleus – assists in export to cytosol Mechanism: Capping enzyme associates with phosphorylated CTD of RNA pol II – capping enzyme specifically associates only w/ RNA pol II transcripts b/c both RNA pol I & III lack phosphorylated CTD CTD:C-terminal domain  Polyadenylation large multi-protein cleavage/polyadenylation complex forms around the poly(A) signals in pre-mRNA – protein-nucleic acid interaction & protein-protein interactions primary pre-mRNA transcript is cleaved by a cleavage factor (endonuclease) at a poly (A) site generating free 3’ OH to which poly(A) polymerase adds up to 250 A residues protects 3’ end of mRNA from enzymatic exonuclease degradation  RNA Splicing Introns are removed and exons are spliced together Intron removal (generally) takes place at every exon/intron junction 1° RNA 5' Intron 3' 5' Intron 3' transcript: 5' 5' 3' 5' Exon 3' 5' 3' 3' Exon Exon 5' 3' Mature mRNA: AAAAAAn m7Gppp Cap  RNA Splicing: Splice sites splice sites: intron cut at exon-intron junctions – both 5’ splice site (5’end of intron) & 3’ splice site (3’end of intron) – location in pre-mRNA determined by comparing genomic DNA and cDNA sequences Fig 10-7 Splicing requires conserved sequences at each end of every intron – So changing or removing central intron region doesn’t affect splicing – Invariant nucleotides include: G-U @ 5’ splice site A @ Branch point (20-50nt away from 3’ splice site) Pyrimidine-rich region A-G @ 3’ splice site-  RNA Splicing Mechanism snRNA: 5 U-rich small nuclear RNAs (U1, U2, U4, U5, U6) that participate in splicing – snRNPs: small nuclear ribonucleoprotein particles are snRNA associated with proteins Spliceosome: large ribonucleoprotein complex of snRNPs that assemble on pre-mRNA and carry out splicing – Assembly: U1 snRNA base pairs with 5’ splice site of the intron U2 interacts with sequence around Branch point A complex of U4/U6/U5 associate RNA Splicing Mechanism cont U1 & U4 released Two transesterification reactions: – circular lariat intron removed – exons ligated debranching enzyme converts lariat intron into linear RNA nuclear exonucleases cut linear intron from both ends – nucleotides recycled What protects the mRNA from the exonucleases??  Trans-Splicing Normally, in most eukaryotes, functional mature mRNAs are derived from processing a single pre-mRNA via cis-splicing Trans-splicing: a special form of RNA processing in eukaryotes where a single mature mRNA is generated from multiple different primary RNA transcripts. – Constructed by ligating together exons of separate RNA molecules – For example, trans-splicing seen in: C. elegans (nematode, round worm) Trypanosomes (cause sleeping sickness and Chagas disease) Euglenoids (freshwater, photosynthetic flagellate)  Alternative Splicing Process by which the same premature mRNA is differently processed in various cells by splicing different exons (of the same gene) resulting in different mature mRNAs – Different combinations of exons from pre-mRNA are joined – Resultant proteins are called isoforms – Regulated by RNA-binding proteins that bind specific sequences near regulated splice sites. Alternate RNA processing pre-mRNAs produced from complex TS units is a significant gene-control mechanism in higher eukaryotes Fibronectin gene contains many exons, but alternative splicing of pre-mRNA varies by cell type.  RNA Editing Exon nucleotides are altered prior to mature mRNA production – pre-mRNA sequence is changed in the nucleus – result: sequence of corresponding mature mRNA differs from the exons encoding it in genomic DNA Quite rare phenomenon in higher eukaryotes – most often: mitochondria of plants & protozoans and in chloroplasts – A mammalian example: RNA editing of apo-B pre-mRNA Apo-B gene encodes serum protein central to cholesterol uptake & transport N-term portion (green) binds lipids, but full length ApoB-100 needed to bind to LDL receptors on cell membranes via the C-term  Ribozymes Ribozymes are RNA molecules with catalytic activity Include: – 23S and 28S rRNA of ribosomes that have peptidyl transferase activity – Group I self-splicing introns of nuclear rRNA genes of protozoans – Group II self-splicing introns of protein-coding genes and some rRNA and tRNA genes in mitochondrial and chloroplast of plant and fungi  Transport Across the Nuclear Membrane Nucleus surrounded by two membranes: each membrane consists of phospholipid bilayer and proteins. Molecules of all sizes enter and leave the nucleus via Nuclear Pore Complexes (NPC) NPCs are large allowing for bidirectional transport: – passive diffusion of small molecules, ions – selective energy- dependent transport of nuclear proteins, RNAs, and RNPs (>9nm)  Nuclear Pore Complex Structure NPC composed of different nucleoporin proteins attached to nuclear basket on nuclear side, cytoplasmic filaments on cytoplasmic side, and central transporter through the middle  FG Nucleoporins and Transporters FG nucleoporins contain many Nuclear transporters short hydrophobic FG repeats and have hydrophobic long hydrophilic regions regions on their surface (dark blue dots) that bind reversibly to FG-domains in FG-nucleoporin. Form molecular sieve that allows H2O-soluble molecules through, but not macromolecules  Import into the Nucleus Proteins synthesized in the cytosol and meant for use in the nucleus enter via the NPC Such proteins contain a nuclear-localization signal (NLS) that directs their translocation into nucleus – NLS is a short amino acid sequence often near the protein’s C-terminus Importins are the transport proteins that bind the NLS and transport the NLS containing cargo into the Immunofluorescence shows fusing a nucleus NLS to a cytoplasmic protein causes the protein to enter the nucleus. Mechanism for Nuclear Import Free cytosolic importin binds NLS of cargo protein Complex moves through NPC, interacting w/ nucleoporins Once in nucleoplasm, conformational change of importin (via interaction w/ Ran-GTP) results in lower affinity for NLS and release of cargo Transporters are recycled  Export out of the Nucleus Similar mechanism used to export proteins, tRNAs and ribosomal subunits from the nucleus to the cytoplasm. Exportin: transport cargo proteins containing nuclear-export signals (NES) out of the nucleus Export mechanism: In nucleoplasm, exportin binds to NES of cargo protein and Ran-GTP Complex diffuses through NPC via interactions w/ nucleoporins Dissociation from complex after Ran-GTP hydrolysis Transporters recycled  Summary: Import & Export of Proteins through NPC Cargo proteins bear an NES or NLS, translocate thru NPC – Some proteins shuttle b/w nucleus & cytoplasm, and must have both NLS & NES Both processes require Ran, a G protein that exists in different conformations when bound to GTP or GDP  GTP Switch Proteins Guanine nucleotide-binding proteins: – switch “on” when GTP bound – switch “off” when GDP bound Signal induced conversion of inactive to active state is mediated by GEF, releasing GDP, allowing GTP to bind – GEF: guanine nt exchange factors Conversion from active to inactive form by hydrolysis of GTP, accelerated by GAP – GAP: GTPase-activating proteins Fig 3-32  mRNP Transport out of Nucleus mRNA exporter protein directs most mRNPs thru nuclear pores – mRNA-exporter diffuses thru the pore, making transient interactions with FG-nucleoproteins as it progresses 3 domains of large subunit: – N bind to mRNP – M bind to mRNP & FG nucleoporins – C bind to FG nucleoporins Small subunit helps M domain binding to FG repeats