Post-transcriptional regulation of gene expression – 1 16-10-2017

INTRODUCTION TO POST-TRANSCRIPTIONAL REGULATION

Post-transcriptional gene regulation After transcription the primary transcript can be regulated at the following stages: It undergoes modifications and extensive processing. 2) Transportation of mature and functional RNAs to the cytoplasm as ribonucleoproteins. 3) Stability of RNA in the cytoplasm. INTRODUCTION TO POST-TRANSCRIPTIONAL REGULATION

Major regulatory control points 1. Splicing INTRODUCTION TO POST-TRANSCRIPTIONAL REGULATION

INTRODUCTION TO POST-TRANSCRIPTIONAL REGULATION The multiple exons of the Drosophila Dscam gene. The Dscam gene (shown at the top) is 61.2 kb long; once transcribed and spliced, it produces one or more versions of a 7.8-kb, 24-exon mRNA (the figure shows the generic structure of those mRNAs). As shown, there are several mutually exclusive alternatives for exons 4, 6, 9, and 17. Thus, each mRNA will contain one of 12 possible alternatives for exon 4 (in red), one of 48 for exon 6 ( purple), one of 33 for exon 9 (green), and one of two for exon 17 (yellow). Exons 4, 6, and 9 encode parts of three Ig domains, depicted in the corresponding colors, and exon 17 encodes the transmembrane domain. If all possible combinations of these exons are used, theDscamgene produces 38,016 different mRNAs and proteins. (Adapted, with permission, from Schmucker D. 2000.Cell 101:671, Fig. 8.#Elsevier.) Molecular Biology of the Gene. 7th edition. Watson, J.D., et al.

Major regulatory control points Splicing Export of RNA INTRODUCTION TO POST-TRANSCRIPTIONAL REGULATION

INTRODUCTION TO POST-TRANSCRIPTIONAL REGULATION mRNA transport mechanism is distinct from other RNA transport mechanisms INTRODUCTION TO POST-TRANSCRIPTIONAL REGULATION Gene Gating – Blobel proposed Exporting RNA from the nucleus to the cytoplasm Nature Reviews, MCB, Volume 8, October 2007

Major regulatory control points Splicing Export of RNAs Localization INTRODUCTION TO POST-TRANSCRIPTIONAL REGULATION

Mature transcripts can be localized through at least three mechanisms: local protection from degradation, diffusion and local anchoring, and direct transport by interaction with the cytoskeleton and cytoskeleton-associated motor proteins FMRP INTRODUCTION TO POST-TRANSCRIPTIONAL REGULATION Regulation of mRNA transport, localization and translation in the nervous system of mammals International Journal of Molecular Medicine April 2014, Volume 33 Issue 4 Translational control at the synapse: role of RNA regulators TIBS, Volume 38, Issue 1, January 2013, Pages 47–55

Major regulatory control points Splicing Export of RNAs Localization Stability Capping Poly-A tail miRNAs INTRODUCTION TO POST-TRANSCRIPTIONAL REGULATION mRNAs for house keeping genes Vs regulatory molecules mRNAs encoding cytokines

Major regulatory control points Splicing Export of RNAs Localization Stability Capping Poly-A tail miRNAs INTRODUCTION TO POST-TRANSCRIPTIONAL REGULATION 5. Translation 6. Protein folding 7. Post-translational modification

Processing of eukaryotic pre-mRNA 5’ capping 3’ cleavage/polyadenylation RNA splicing INTRODUCTION TO POST-TRANSCRIPTIONAL REGULATION

INTRODUCTION TO POST-TRANSCRIPTIONAL REGULATION

mRNA processing is tightly coupled to transcription. The phosphorylation state of the C-terminal domain (CTD) of RNA Polymerase II (Pol II) is given on the right in relation to major steps of transcription. Listed are functional processing sequences (red), components of processing machinery (blue) and factors that are loaded onto the transcript as a result of processing (green). INTRODUCTION TO POST-TRANSCRIPTIONAL REGULATION

RNA binding proteins play major roles in RNA metabolism INTRODUCTION TO RNA BINDING PROTEINS

Some RNA binding proteins can shuttle in and out of the nucleus whereas others cannot hnRNP C hnRNP A1 HeLa cell Unfused Xenopus cell Fused heterokaryon INTRODUCTION TO RNA BINDING PROTEINS

CAPPING The structure and formation of the 5’ RNA cap. In the first step, the γ-phosphate at the 5’ end of the RNA is removed by an enzyme called RNA triphosphatase (the initiating nucleotide of a transcript initially retains its α-, β-, and γ-phosphates). In the next step, the enzyme guanylyltransferase adds a GMP moiety to the resulting terminal β-phosphatase. This is a two-step process: first, an enzyme–GMP complex is generated from GTP with release of the β-and γ- phosphates of that GTP, and then the GMP from the enzyme is transferred to the β-phosphate of the 5’ end of the RNA. Once this linkage is made, the newly added guanine and the purine at the original 5’ end of the mRNA are further modified by the addition of methyl groups by methyltransferase. The resulting 5’ cap structure subsequently recruits the ribosome to the mRNA for translation to begin Molecular Biology of the Gene. 7th edition. Watson, J.D., et al.

5’ capping After nascent RNA molecules reach a length of 25-30 nucleotides a 7 methyl guanosine residue is added to the 5’ end creating a 5’-5’ triphosphate structure. The capping enzyme is associated with the phosphorylated CTD of RNA pol II. 5’ cap structure interacts with the CAP-binding complex made up of Cap-binding proteins. The function of the 5’ cap is to Identify the 5’ end of the RNA Involved in translocation of mRNA to cytoplasm Protection of the mRNA from degradation 5’CAP interacts with translation initiation factors

SPLICING

SPLICING Molecular Biology of the Gene. 7th edition. Watson, J.D., et al.

SPLICING Molecular Biology of the Gene. 7th edition. Watson, J.D., et al.

Splicing During formation of a mature, functional mRNA, the introns are removed and exons are spliced together The location of splice sites in a pre mRNA can be identified by comparing sequence of genomic DNA to a cDNA prepared from the corresponding mRNA. SPLICING Three areas are 100% conserved “GU” at 5’ splice site in the intron “AG” at 3’ splice site in the intron “A” at branch point in the intron

Splicing occurs through two sequential transesterification reactions Molecular Biology of the Gene. 7th edition. Watson, J.D., et al.

snRNAs base-pair with pre-mRNA and with one another during splicing Five U-rich small nuclear RNAs (snRNAs) designated U1, U2, U4, U5 and U6 participate in mRNA splicing. These RNAs range in length from 107 to 210 nucleotides and are associated with 6- 10 proteins in small nuclear ribonucleoprotein particles. A total of ~170 proteins participate in splicing along with the five snRNAs. Spliceosome is as big as ribosomes. SPLICING Two points: (1) The sequence surrounding the 5’splice DONOR site or the branch point “A” or the 3’splice acceptor site are not conserved (please see previous slide). How come then splicing happens in such pre-mRNAs? Suggests that there is something more than sequence of the pre-mRNA which may be critical – structure??? (2) This is reinforced by the fact that mutation in non-conserved flanking sequence ALSO affects splicing (see the fig above). SEE THE SLIDE ABOUT SR PROTEINS – 5th slide from this one.

Spliceosomes, assembled from snRNPs and a pre-mRNA carry out splicing A spliceosome is roughly the size of a ribosome and is composed of about 170 proteins, including about 100 “splicing factors” in addition to the proteins associated with the five snRNPs. SPLICING FIGURE 8-11 Model of spliceosome-mediated splicing of pre-mRNA. Step 1 : After U1 base pairs with the consensus 5‘ splice site, SF1 (splicing factor 1) binds the branch point A; U2AF (U2 snRNP associated factor) associates with the polypyrimidine tract and 3'splice site; and the U2 snRNP associates with the branch point A via base pairing interactions shown in Figure 8-9, displacing SF1 . Step 2: A trimeric snRNP complex of U4, US, and U6 joins the initial complex to form the spliccosome. Step 3: Rearrangments of base-pairing interactions between snRNAs converts the spliceosome into a catalytically active conformation and destabilizes the U1 and U4 snRNPs, which are released. Step 4: The catalytic core, thought to be formed by U6 and U2, then catalyzes the first transesterification reaction, forming the intermediate containing a 2'.5'-phosphodiester bond as shown in Figure 8-8. Step 5: Following further rearrangements between the snRNPs, the second transesterification reaction joins the two exons by a standard 3 ',S '-phosphodiester bond and releases the intron as a lariat structure and the remaining snRNPs. Step 6: The excised lariat intron is converted into a linear RNA by a debranching enzyme. [Adapted from T. Villa et al .. 2002, Ce// 109:149.]

SPLICING

CTD as an integration platform for different RNA processing steps SPLICING Phosphorylated CTD helps in the recruitment of many of the spliceosome components. In turn these hnRNPs enhance the interaction of RNAP II with elongation factors such as DSIF and CDK9-cyclin T (P-TEFb) which increases the rate of transcription elongation.

mRNA processing is tightly coupled to transcription. The phosphorylation state of the C-terminal domain (CTD) of RNA Polymerase II (Pol II) is given on the right in relation to major steps of transcription. Listed are functional processing sequences (red), components of processing machinery (blue) and factors that are loaded onto the transcript as a result of processing (green). INTRODUCTION TO POST-TRANSCRIPTIONAL REGULATION

SPLICING SR proteins recruit spliceosome components to the 5’ and 3‘ splice sites. Legitimate splice sites are recognized by the splicing machinery by virtue of being close to exons. Thus, SR proteins bind to sequences within the exons (exonic splicing enhancers, ESEs), and from there recruit U2AF and U1snRNP to the downstream 5’ and upstream 3’ splice sites, respectively—a process often called exon definition. This initiates the assembly of the splicing machinery on the correct sites, and splicing can proceed as outlined above. (Adapted, with permission, from Maniatis T. and Tasic B. 2002.Nature418:236–243.#Macmillan.) Molecular Biology of the Gene. 7th edition. Watson, J.D., et al.

How SR proteins discriminate exons from introns? Not known More or else uniform size of the exons help ESEs help Bruce Alberts, Molecular Biology, 5th Edition

Splicing is carried out by ribozymes

3’ cleavage and polyadenylation are tightly linked Two sequences at the 3’-end of the RNA determine the site of cleavage and Polyadenylation. 1) AAUAAA sequence present 10-35 Nucleotides upstream of the Poly A Site. 2) GU-rich or U rich sequence present ~ 50 nucleotide downstream of the Poly A site. A large multiprotein complex assembles at the cleavage/ polyadenylation site to carry out this process. First CPSF(clevage and polyadenylation specific factor) recognizes and binds to AAUAAA sequence Followed by CStf (cleavage stimulatory factor) binding to the G/U rich site This is followed by CFI and CFII binding Finally Poly (A) polymerase (PAP) binds. Cleavage occurs first

Cleavage is closely Followed by polyadenylation Which occurs in two steps The first 12 or so A residues are added by PAP at a very slow rate. 2) This is followed by a rapid addition of 250 or so As by PAP but this time it is stimulated by binding of PABPII (poly A binding protein II). PAPBII also signals PAP to stop When approximately 200-250 A residues have been added. The mechanism for controlling the length of the poly A tail is not well understood.

A Unified Theory of Gene Expression- George Orphanides and Danny Reinberg, Volume 108, Issue 4, 22 February 2002, Pages 439-451

Animations: https://www.dnalc.org/view/16938-3D-Animation-of-RNA-Splicing.html https://www.youtube.com/watch?v=YgmoHtLGb5c