Chapter 10 - Post-transcriptional Gene Control

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1 Chapter 10 - Post-transcriptional Gene Control

2 Chapter 10 - Post-transcriptional Gene Control 10.1 Processing of Eukaryotic Pre-mRNA 10.2 Regulation of Pre-mRNA Processing 10.3 Transport of mrna Across the Nuclear Envelope 10.4 Cytoplasmic Mechanisms of Post-transcriptional Control

3 Post-transcriptional Gene Control 10.1 Processing of Eukaryotic Pre-mRNA Pre-mRNA is capped, polyadenylated, spliced, and associated with RNPs in the nucleus before export to ribonucleoprotein the cytoplasm.

4 Overview of RNA processing and post-transcriptional gene control. Step 1 Use of alternative exons during pre-mrna splicing Step 2 Use of alternative poly(a) sites. Step 3 Properly processed mrnas exported to the cytoplasm Improperly processed mrnas blocked from export to the cytoplasm degraded the exosome complex containing multiple ribonucleases Step 4 Translation initiation factors bind to the 5 cap cooperatively with poly(a)-binding protein I bound to the poly(a) tail and initiate translation Step 5 mrna degraded in cytoplasmic P bodies translational repression Deadenylated and decapped by enzymes Degraded by cytoplasmic exosomes Control of mrna degradation rate regulates mrna abundance and amount of protein translated Step 6 mrnas synthesized without long poly(a) tails translation regulated by controlled synthesis of a long poly(a) tail by a cytoplasmic poly(a) polymerase Step 7 Translation regulation by other mechanisms mirna (~22-nucleotide RNAs) inhibit translation of mrnas to which they hybridize, usually in the 3 untranslated region Step 8a trnas transcribed by Pol III and processed in the nucleus Step 8b rrnas transcribed by Pol I processed in the nucleolus Step 9 Regions of precursors cleaved from the mature RNAs degraded by nuclear exosomes

5 Overview of mrna processing in eukaryotes Why cleavage? Step 1: Nascent RNA (β-globin RNA) 5 end capped with 7-methylguanylate (shortly after RNA polymerase II initiates transcription at the first nucleotide of the first exon of a gene) Pol II transcription terminates at any one of multiple termination sites downstream from the poly(a) site in final exon Step 2: Cleavage enzyme cleaves primary transcript at the poly(a) site. Step 3: Polyadenylation enzyme adds a string of adenosine (A) residues (~250 A residues in mammals, ~150 in insects, and ~100 in yeast). Step 4: Short primary transcripts with few introns splicing follows cleavage and polyadenylation (shown) Long transcripts with multiple introns introns spliced out of the nascent RNA during transcription

6 Cellular RNAs and precursors.

7 Splicing process

8 Electron microscopy of mrna template DNA hybrids shows that introns are spliced out during pre-mrna processing.

9 Consensus sequences around splice sites in vertebrate pre-mrnas. Intron splice site invariant bases (flanking bases indicated found at frequencies higher than expected for a random distribution): 5 GU 3 AG Branch-point adenosine usually bases from the 3 splice site Polypyrimidine tract near the 3 end of the intron found in most introns Central region: 40 bases 50 kilobases Only nucleotides at each end of an intron are necessary for splicing to occur at normal rates.

10 transesterification reactions result in the splicing of exons in pre-mrna. Exon splicing two sequential transesterification reactions [Arrows indicate where activated hydroxyl oxygens react with phosphorus atoms]: Reaction 1 intron 5 phosphorusexon one 3 oxygen ester bond exchanged for an intron 5 phosphorus ester bond with the branch-point A residue 2 oxygen Reaction 2 the exon two 5 phosphorus intron 3 oxygen ester bond exchanged for an exon two 5 phosphorus ester bond with the 3 oxygen of exon one joins the two exons releases intron as a lariat structure How??

11 Base pairing between pre-mrna, U1 snrna, and U2 snrna early in the splicing process. Five U-rich snrnas U1, U2, U4, U5, and U6 ( nucleotides long) participate in premrna splicing. Base-pair with pre-mrna Interact with 6 10 proteins each form small nuclear ribonucleoprotein particles (snrnps)

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13 mrna splicing Two actions cut and ligation

14 mrnp From their transcriptional birth to their degradation, cellular mrnas are coated with proteins in messenger ribonucleoprotein (mrnp) complexes. The mrnp composition controls every aspect of the life of the mrna, from pre-mrna processing to mrna localization, translation and turnover.

15 GU----A-----AG? How to recognize the exon and intron?

16 SR proteins SR proteins are a conserved family of proteins involved in RNA splicing. SR proteins are named because they contain a protein domain with long repeats of serine and arginine amino acid residues, whose standard abbreviations are "S" and "R" respectively.

17 Exon recognition through cooperative binding of SR proteins and splicing factors to pre-mrna. SR proteins contribute to exon definition in long pre-mrnas. SR proteins: Interact with exonic enhancer sequences Pre-mRNAs (humans): (ESEs) within exons Exons avg ~150 bases Contain several RS protein-protein Introns avg ~3500 bases longest exceed interaction domains rich in arginine (R) and 500 kb serine (S) residues Degenerate 5 and 3 splice site and branch Mediate cooperative binding of U1 snrnp to point sequences multiple copies likely to a true 5 splice site and U2 snrnp to a branch occur randomly in long introns point through a network of protein-protein Additional sequence information is interactions that span an exon required to define the exons that should be spliced together in higher organism premrnas with long introns.

18 Self-splicing introns Self-splicing introns two types: Group I introns in nuclear rrna genes of protozoans Group II introns In protein-coding genes and some rrna and trna genes in mitochondria and chloroplasts of plants and fungi Fold into a conserved, complex secondary structure containing numerous stem-loops

19 Post-transcriptional Gene Control 10.2 Regulation of Pre-mRNA Processing Alternative promoters, primary transcript alternative splicing, and cleavage at different poly(a) site cleavages yield different mrnas from the same gene in different cell types or at different developmental stages. RNA-binding proteins that bind to specific sequences near splice sites regulate alternative splicing. Rare RNA editing of mrna sequences in the nucleus have important consequences by altering the amino acid encoded by an edited codon.

20 A cascade of regulated splicing controls sex determination in Drosophila embryos. (a) Sex-lethal (Sxl) protein: intronic splicing silencer Present only in female embryos (no functional Sxl protein expressed in males) Binds to a premrna sequence near the 3 end of the intron between exon 2 and exon 3 blocks U2AF and U2 snrnp association of with the adjacent 3 splice site (used in males). female splicing (exon 2 4) deletes exon 3 (contains premature stop codon) Sxl expressed male splicing (exon 2 3) mrna contains exon 3 premature stop codon. no functional Sxl protein expressed in males (b) Sxl blocks tra exon 1 2 splicing. Only female embryos produce functional Tra protein. Tra promotes specific Dax splicing (c) Dsx (transcription factor) expression: Females cooperative binding of Tra protein and Rbp1 and Tra2 SR proteins activates exon 3 4 splicing and cleavage/polyadenylation(an) at the 3 end of exon 4. expresses female Dsx isoform with exon 4-encoded sequence transcription factor Males embryos lack functional Tra Series of different proteins are expressed! SR proteins do not bind to exon 4 exon 3 is spliced to exon 5. expresses male Dsx isoform with exon 5-encoded sequence (transcription repressor) Distinct female and male Dsx proteins: female Dsx isoform activates genes with Dsx transcription factor binding sites, including genes that induce development of female characteristics male Dsx protein represses expression of the same target genes with Dsx binding sites

21 Model of splicing activation by Tra protein and the SR proteins Rbp1 and Tra2. One of the SR proteins

22 Role of alternative splicing in the perception of sounds of different frequencies. Frequencies Ca 2+ -activated K + channel Channel isoforms encoded by alternatively spliced mrnas produced from the same primary transcript alternative exons used at eight regions in the mrna 576 possible isoforms [Red numbers regions where alternative splicing produces different isoforms] respond to different frequencies by opening at different Ca 2+ concentrations

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24 Overview of RNA processing and post-transcriptional gene control. Next?

25 Post-transcriptional Gene Control 10.3 Transport of mrna Across the Nuclear Envelope An mrnp exporter ensures directional export by binding mrnps in the nucleus, facilitating transport across the NPC, and releasing the mrnps when mrnp adapter proteins are phosphorylated in the cytoplasm. The mrnp exporter binds most mrnas cooperatively with SR proteins bound to exonic splicing enhancers and with REF associated with exon-junction complexes as well as with additional mrnp proteins. Pre-mRNAs still bound to spliceosomes are not exported, ensuring only mature mrnas reach the cytoplasm.

26 Remodeling of mrnps during nuclear export. Some mrnp proteins dissociate from nuclear mrnp complexes before export through an NPC. Some mrnps remain associated CBC (cap binding complex), NXF1/NXT1, and PABPN1 bound to the poly(a) tail exported with the mrnp dissociate from the mrnp in the cytoplasm and are shuttled back into the nucleus through an NPC Translation initiation factor eif4e replaces CBC bound to the 5 cap. PABPC1 replaces PABPN1.

27 Nuclear pore complex (NPC)

28 Nuclear export

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30 Formation of heterogeneous ribonucleoprotein particles (hnrnps) and export of mrnps from the nucleus.

31 Poly-adenylation

32 Model for cleavage and polyadenylation of pre-mrnas in mammalian cells. Mechanism: CPSF (Cleavage and polyadenylation specificity factor) binds to the upstream AAUAAA polyadenylation signal CStF interacts with a downstream GU- or U-rich sequence and with bound CPSF, forming a loop in the RNA. CFI and CFII binding stabilize the complex PAP (Poly(A) polymerase) stimulates cleavage at a poly(a) cleavage site (typically nucleotides 3 of the upstream polyadenylation signal) Cleavage factors released Downstream RNA cleavage product rapidly degraded PAP adds ~12 A residues (from ATP) at a slow rate to the 3 -hydroxyl group generated by the cleavage reaction PABPN1 (Nuclear poly(a)-binding protein) Binds to the initial short poly(a) tail Accelerates the rate of addition by PAP Signals PAP to stop adding As after A residues have been added

33 Post-transcriptional Gene Control 10.4 Cytoplasmic Mechanisms of Post-transcriptional Control Stability of most mrnas is controlled by poly(a) tail length and binding of various proteins to 3 UTR sequences. mrna translation can be regulated by micro-rnas and RNA interference by sirnas and various degradation, cytoplasmic splicing, and polyadenylation mechanisms. Many mrnas are transported to specific subcellular locations by sequence-specific RNA-binding proteins that bind 3 UTR localization sequences.

34 Pathways for degradation of eukaryotic mrnas. most common pathway Deadenylase complex shortens poly(a) tail to 20 A residues. Destabilizes PABPC1 binding PABC1 loss weakens interactions between the 5 cap and translation initiation factors. Deadenylated mrna:

35 P body Processing bodies (P-bodies) are distinct foci within the cytoplasm of the eukaryotic cell consisting of many enzymes involved in mrna turnover. P-bodies have been observed in somatic cells originating from vertebrates and invertebrates, plants and yeast. To date, P-bodies have been demonstrated to play fundamental roles in general mrna decay, nonsensemediated mrna decay, adenylate-uridylate-rich element mediated mrna decay, and microrna induced mrna silencing. 1. decapping and degradation of unwanted mrnas 2. storing mrna until needed for translation 3. aiding in translational repression by mirnas (related to sirnas)

36 (a) mirnas: Repress translation of target mrnas Hybridize imperfectly with target mrnas mirna nucleotides 2 7 (seed sequence) most critical for targeting it to a specific mrna 3 -UTR (b) sirna: Hybridizes perfectly with target mrna Causes cleavage of mrna (position indicated by the red arrow), triggering its rapid degradation

37 RISC The RNA-induced silencing complex, or RISC, is a multiprotein complex, specifically a ribonucleoprotein, which incorporates one strand of a single-stranded RNA (ssrna) fragment, such as microrna (mirna), or double-stranded small interfering RNA (sirna).[1] The single strand acts as a template for RISC to recognize complementary messenger RNA (mrna) transcript. Once found, one of the proteins in RISC, called Argonaute, activates and cleaves the mrna. This process is called RNA interference (RNAi) and it is found in many eukaryotes; it is a key process in gene silencing and defense against viral infections

38 Processing of mirna mirna transcription and processing: RNA polymerase II transcribes primary mirna transcripts (primirna) folds to form double strand region Nuclear double-strand RNA specific endoribonuclease Drosha and double-strand RNA binding protein DGCR8 (Pasha in Drosophila) bind pri-mirna double strand regions Drosha cleaves the pri-mirna generates a ~70-nucleotide premirna Exportin 5 nuclear transporter transports processed pri-mirna to the cytoplasm Dicer in conjunction with the double-stranded RNA binding protein TRBP (Loquacious in Drosophila) processes pre-mirna into a double-stranded mirna with a two-base single-stranded 3 end RISC complex binds one of the two strands. incorporates mature mirna into complex with Argonaute proteins mrna translation inhibition: mirna-risc complexes associate with target mrnps by base pairing between the Argonaute-bound mature mirna and complementary regions in the 3 UTRs of target mrnas The more RISC complexes bound to the 3 UTR of an mrna, the greater the repression of translation RISC complex binding causes bound mrnps to associate with P bodies mrna degraded Alternative polyadenylation increases mirna control options.

39 The function of mirnas in limb development. Result fundamental pattern maintained but abnormal limb development Conclusion Dicer (mirna) is required for morphogenesis but not patterning of the vertebrate limb

40 Polyadenylation and translation initiation

41 Model for control of cytoplasmic polyadenylation and translation initiation. Immature oocyte mrnas containing the U- rich cytoplasmic polyadenylation element (CPE) initially have short poly(a) tails translation repression: Hormonal stimulation of oocyte maturation: Activates a protein kinase that phosphorylates CPEB Phosphorylated CPEB releases Maskin recruits CPSF (cleavage and polyadenylation specificity factor) binds the poly(a) site CPSF recruits the cytoplasmic poly(a) polymerase (PAP). PAP lengthens the poly(a) tail. Longer poly(a) tail binds multiple copies of cytoplasmic poly(a)-binding protein 1 (PABPC1). PABC1 interacts with eif4g stabilizes interaction of factors for ribosome recruitment and translation

42 Discussion with friends Find what is the nuclear localization sequence (NLS) of protein and its mechanism Search the Clustered regularly interspaced short palindromic repeats in the wikipedia site and explain the right figure. ( CRISPR). Transgenic monkey (2014, Nature)

43 Discussion with friends Find this interesting paper!!! What are the autosome and X chromosome? How does the male drosophila have no sexlethal protein? Explain the right figure 2