1. Post-transcriptional gene control

2. Subjects, covered in the lecture Processing of eukaryotic pre-mRNA - capping -polyadenylation -splicing -editing Nuclear transport

4. Processing of eukaryotic pre-mRNA: the classical texbook picture

5. Alternative picture: co-transcriptional pre-mRNA processing This picture is more realistic than the previous one, particularly for long pre-mRNAs

6. Heterogenous ribonucleoprotein patricles (hnRNP) proteins In nucleus nascent RNA transcripts are associated with abundant set of proteins hnRNPs prevent formation of secondary structures within pre-mRNAs hnRNP proteins are multidomain with one or more RNA binding domains and at least one domain for interaction with other proteins some hnRNPs contribute to pre-mRNA recognition by RNA processing enzymes The two most common RNA binding domains are RNA recognition motifs (RRMs) and RGG box (five Arg-Gly-Gly repeats interspersed with aromatic residues)

7. 3D structures of RNA recognition motif (RRM ) domains

8. Capping p-p-p-N-p-N-p-N-p…. p-p-N-p-N-p-N-p… G-p-p-p-N-p-N-p-N-p… CH 3 G-p-p-p-N-p-N-p-N-p… CH 3 CH 3 GMP mCE (another subunit) Capping enzyme (mCE) methyltransferases S-adenosyl methionine

9. The capping enzyme A bifunctional enzyme with both 5’-triphosphotase and guanyltransferase activities In yeast the capping enzyme is a heterodimer In metazoans the capping enzyme is monomeric with two catalytic domains The capping enzyme specific only for RNAs, transcribed by RNA Pol II (why?)

10. Capping mechanism in mammals DNA Growing RNA Capping enzyme is allosterically controlled by CTD domains of RNA Pol II and another stimulatory factor hSpt5

11. Polyadenylation Poly(A) signal recognition Cleavage at Poly(A) site Slow polyadenylation Rapid polyadenylation

12. G/U: G/U or U rich region CPSF: cleavage and polyadenylation specificity factor CStF: cleavage stimulatory factor CFI: cleavage factor I CFII: cleavage factor II

13. PAP: Poly(A) polymerase

15. CPSF PAP

16. PABPII- poly(A) binding protein II

17. PABP II functions: 1.rapid polyadenylation 2.polyadenylation termination

18. p p Pol II ctdctd mRNA PolyA – binding factors Link between polyadenylation and transcription Pol II gets recycled mRNA gets cleaved and polyadenylated degradation cap polyA cap splicing, nuclear transport p p aataaa FCP1 Phosphatase removes phospates from CTDs cap

19. Splicing

20. The size distribution of exons and introns in human, Drosophila and C. elegans genomes

21. Consensus sequences around the splice site YYYY

22. Molecular mechanism of splicing

23. Small nuclear RNAs U1-U6 participate in splicing snRNAs U1, U2, U4, U5 and U6 form complexes with 6-10 proteins each, forming small nuclear ribonucleoprotein particles (snRNPs) Sm- binding sites for snRNP proteins

24. The secondary structure of snRNAs

25. Additional factors of exon recognition ESE - exon splicing enhancer sequences SR – ESE binding proteins U2AF65/35 – subunits of U2AF factor, binding to pyrimidine-rich regions and 3’ splice site

26. Binding of U1 and U2 snRNPs Binding of U4, U5 and U6 snRNPs The essential steps in splicing

27. Rearrangement of base-pair interactions between snRNAs, release of U1 and U4 snRNPs

28. The catalytic core, formed by U2 and U6 snRNPs catalyzes the first transesterification reaction

29. Further rearrangements between U2, U6 and U5 lead to second transesterification reaction

30. The spliced lariat is linearized by debranching enzyme and further degraded in exosomes Not all intrones are completely degraded. Some end up as functional RNAs, different from mRNA

31. p p Pol II ctdctd mRNA SCAFs: SR- like CTD – associated factors cap SRs snRNPs Intron Co-transciptional splicing

32. Self-splicing introns Under certain nonphysiological conditions in vitro, some introns can get spliced without aid of any proteins or other RNAs Group I self-splicing introns occur in rRNA genes of protozoans Group II self-splicing introns occur in chloroplasts and mitochondria of plants and fungi

33. Group I introns utilize guanosine cofactor, which is not part of RNA chain

34. Comparison of secondary structures of group II self- splicing introns and snRNAs

35. Spliceosome Spliceosome contains snRNAs, snRNPs and many other proteins, totally about 300 subunits. This makes it the most complicted macromolecular machine known to date. But why is spliceosome so extremely complicated if it only catalyzes such a straightforward reaction as an intron deletion? Even more, it seems that some introns are capable to excise themselves without aid of any protein, so why have all those 300 subunits?

36. No one knows for sure, but there might be at least 4 reasons: 1. Defective mRNAs cause a lot of problems for cells, so some subunits might assure correct splicing and error correction 2. Splicing is coupled to nuclear transport, this requires accessory proteins 3. Splicing is coupled to transcription and this might require more additional accessory proteins 4. Many genes can be spliced in several alternative ways, which also might require additional factors

37. One gene – several proteins Cleavage at alternative poly(A) sites Alternative promoters Alternative splicing of different exons RNA editing

38. Alternative splicing, promoters & poly-A cleavage

39. RNA editing Enzymatic altering of pre-mRNA sequence Common in mitochondria of protozoans and plants and chloroplasts, where more than 50% of bases can be altered Much rarer in higher eukaryotes Editing of human apoB pre-mRNA

40. The two types of editing 1) Substitution editing Chemical altering of individual nucleotides Examples: Deamination of C to U or A to I (inosine, read as G by ribosome) 2) Insertion/deletion editing Deletion/insertion of nucleotides (mostly uridines) For this process, special guide RNAs (gRNAs) are required

41. Guide RNAs (gRNAs) are required for editing

42. Organization of pre-rRNA genes in eukaryotes

43. Electron micrograph of tandem pre-rRNA genes

44. Small nucleolar RNAs ~150 different nucleolus restricted RNA species snoRNAs are associated with proteins, forming small nucleolar ribonucleoprotein particles (snoRNPs) The main three classes of snoRNPs are envolved in following processes: a)removing introns from pre-rRNA b)methylation of 2’ OH groups at specific sites c)converting of uridine to pseudouridine

45. What is this pseudouridine good for? Pseudouridine Y is found in RNAs that have a tertiary structure that is important for their function, like rRNAs, tRNAs, snRNAs and snoRNAs The main role of Y and other modifications appears to be the maintenance of three-dimensional structural integrity in RNAs Uridine ( U ) Pseudouridine ( Y )

46. Where do snoRNAs come from? Some are produced from their own promoters by RNA pol II or III The majority of snoRNAs come from introns of genes, which encode proteins involved in ribosome synthesis or translation Some snoRNAs come from intrones of genes, which encode nonfuctional mRNAs

47. Assembly of ribosomes

48. Processing of pre-tRNAs RNase P cleavage site

49. Splicing of pre-tRNAs is different from pre-mRNAs and pre-rRNAs The splicing of pre-tRNAs is catalyzed by protein only A pre-tRNA intron is excised in one step, not by two transesterification reactions Hydrolysis of GTP and ATP is required to join the two RNA halves

50. Macromolecular transport across the nuclear envelope

51. The central channel Small metabolites, ions and globular proteins up to ~60 kDa can diffuse freely through the channel Large proteins and ribonucleoprotein complexes (including mRNAs) are selectively transported with the assistance of transporter proteins

52. Two different kinds of nuclear location sequences basic hydrophobic importin a importin b nuclear import Proteins which are transported into nucleus contain nuclear location sequences

53. Artifical fusion of a nuclear localization signal to a cytoplasmatic protein causes its import to nucleus

54. Mechanism for nuclear “import”

55. Mechanism for nuclear “export”

56. Mechanism for mRNA transport to cytoplasm

57. Example of regulation at nuclear transport level: HIV mRNAs

58. After mRNA reaches the cytoplasm... mRNA exporter, mRNP proteins, nuclear cap- binding complex and nuclear poly-A binding proteins dissociate from mRNA and gets back to nucleus 5’ cap binds to translation factor eIF4E Cytoplasmic poly-A binding protein (PABPI) binds to poly-A tail Translation factor eIF4G binds to both eIF4E and PABPI, thus linking together 5’ and 3’ ends of mRNA