1. Multiple Steps in Gene Expression after Transcription 1.Transcription 2.5’ Capping 3.3' maturation: cleavage & polyadenylation 4.Splicing 5.Transport of RNA to Cytoplasm 6.Stabilization/Destabilization of mRNA 7.Translation

2. Relative cellular RNA abundance Ribosomal RNAs (rRNAs) ~ 90% Transfer RNAs (tRNAs) ~ 5% Messenger RNAs (mRNAs) ~ 2% The rest (~3%): Signal recognition particle (SRP) RNA Small nuclear RNAs (snRNAs) Small nucleolar RNAs (snoRNAs) Micro RNAs (miRNAs)

3. Likely order of events in producing a mature mRNA from a pre-mRNA.

4. RNA Capping 1.Post-transcriptional (i.e., G not encoded) 2.Involves adding a 7 Me Guanosine nt to the first (RNA) nt in an unusual way, and often methylation of the first few nt of the RNA. 3.Occurs before the pre-mRNA is 30 nt long

5. Fig. 15.3

6. Fig. 15.4 AdoMet = S-adenosylmethionine, the methyl donor Capping: order of events and enzymes Product is Cap 1 or “RNA triphosphatase”

7. Cap Functions Cap provides: 1.Protection from some ribonucleases* 2.Enhanced translation* 3.Enhanced transport from nucleus 4.Enhanced splicing of first intron for some pre-mRNAs * Also functions of the polyA-tail

8. Post-transcriptional Processes II: Pre-mRNA Polyadenylation Most cytoplasmic mRNAs have a polyA tail (3’ end) of 50-250 Adenylates –a notable exception is histone mRNAs Discovered in 1971 (J. Darnell et al.) Added post-transcriptionally by an enzyme, polyA polymerase(s) Turns over (recycles) in cytoplasm

9. Functions of the PolyA-Tail 1.Promotes mRNA stability - De-adenylation (tail shortening) can trigger rapid degradation of the RNA 2.Enhances translation - promotes recruitment by ribosomes - bound by a polyA-binding protein in the cytoplasm, PAB1 - synergistic stimulation with Cap!

10. Methods: The firefly-luciferase mRNAs (with the 5’ and 3’ UTRs from a plant gene) were electroporated into protoplasts. Then, luciferase mRNA, and luciferase activity levels were measured at select times. (from D. Gallie)

11. An Unexpected Mechanism for Polyadenylation of pre-mRNA 1.Transcription extends beyond mRNA end 2.Transcript is cut at 3’ end of what will become the mRNA (in green) 3.PolyA Polymerase adds ~250 As to 3’ end 4.“Extra” RNA (in red) degraded FigFig Fig. 15.12

12. Evidence for Transcription Past the End of a Cellular mRNA Nuclear run-on transcription assay using Friend erythroleukemic cells treated with DMSO (stimulates globin gene transcription) –Newly synthesized RNAs are hybridized to DNA regions that span the globin gene, including regions downstream –The amount of newly synthesized RNA complementary to each gene region is thus quantified

13. Run-on transcription assay with isolated nuclei Fig. 5.33 Only gene Y is transcribed.

14. Transcription beyond the polyA site. After run-on transcription (in the presence of 32 P-UTP) with nuclei from Friend Erythroleukemic cells, the labeled RNA was hybridized to DNA fragments A-F that span the globin gene. The relative molarities of newly synthesized RNA that hybridized to each fragment are given. s.d. is the standard deviation. Notice that there is just as much RNA transcribed from just downstream of the gene (fragment E) as there is from the gene itself (fragments B-D). Fig. 15.14

15. Polyadenylation (PolyA) Signal AAUAAA (in mammals and plants) –Located ~20-30 bp from the polyA-tail –There are other hexamers that will provide this function, but less efficiently. Mutagenesis and in vivo expression studies revealed 2 other motifs downstream of AAUAAA that promote 3’ processing. 1.GU-rich stretch 2.U-rich stretch

16. What if there are multiple possible signals? Competition experiment: 1. The synthetic polyadenylation site (SPA) below was inserted downstream of the normal one in a globin gene (giving a globin gene with 2 signals). Fig. 15.21, 2 ed. GU-rich U-rich* * Absent from the globin gene

17. 2. The globin gene with two 3’ processing signals was introduced into HeLa cells, and the 3’ end of the mRNA determined by S1 mapping. Result: SPA was mainly used for polyAdenylation Conclusion The stronger set of elements (signals) in the SPA outcompeted the native 3’-processing signal, which lacks the U-rich motif.

18. Polyadenylation: The Proteins Proteins required in mammals for cleavage and polyadenylation of a new transcript. Proteins required for efficient cleavage of pre-mRNA: 1.CPSF (cleavage & polyadenylation specificity factor), binds the AAUAAA 2.CstF (cleavage stimulation factor) binds to the G/U rich region cooperatively with CPSF 3.CFI and CFII (cleavage factors I and II), RNA-binding proteins 4.PAP (polyA polymerase) 5.nRNAP II (the CTD of the very large RPB1 subunit) stimulates cleavage James Manley

19. Model for the pre-cleavage complex Fig. 15.19

20. Polyadenylation: Mechanism Occurs in 2 phases –Phase 1: requires AAUAAA and ~8 nt downstream (3’) –Phase 2 : Once ~10 As are added, further adenylation does not require the AAUAAA

21. 2 Phases to polyadenylation Fig. 15.21 Substrates : 1. 58-nt RNA from SV40 that ends with AAUAAA plus 8 nt ( ). 2. Same as (1), but with a 40-nt polyA-tail (A 40 ). 3. Same as (1), but with a random 40-nt at the 3’ end (X 40 ). The series with an X contain a mutated AAUAAA (AAGAAA). Hela-cell nuclear extract was incubated with radioactively-labeled RNA substrates, which were then separated by gel electrophoresis. Conclusion: the AAUAAA not needed for phase 2, only phase 1.

22. Proteins Required for Polyadenylation Phase I: 1.CPSF 2.PolyA polymerase (PAP II) Phase II: 1.PolyA polymerase (PAP II) 2.PolyA Binding Protein II (PAB II) - PAB II binds to short A-tail - Helps PAP II synthesize long tails

23. Nuclear PolyA Polymerase (long form = PAP II) RBD - RNA binding domain NLS - nuclear localization signal PM - polymerase module S/T- serine/threonine rich regions (yellow) Fig. 15.27,3 rd ed.

24. CPSF specificity factor CFI and CFII PAP II CstF stimulation factor PAB II Fig. 15.25