1. RNA PROCESSING AND RNPs

2. RNA Processing  Very few RNA molecules are transcribed directly into the final mature RNA.  Most newly transcribed RNA molecules (primary transcripts) undergo various alterations to yield the mature product  RNA processing is the collective term used to describe the molecular events allowing the primary transcripts to become the mature RNA.

3. primary transcript mature RNA. Nucleus or Nucleolus Cytoplasm RNA processing Romoval of nucleotides addition of nucleotides to the 5’- or 3’- ends modification of certain nucleotides

4. (1) Removal of nucleotides by both endonucleases and exonucleases  endonucleases to cut at specific sites within a precursor RNA  exonucleases to trim the ends of a precursor RNA  This general process is seen in prokaryotes and eukaryotes for all types of RNA

5. (2) Addition of nucleotides to 5 ’ -or 3 ’ - ends of the primary transcripts or their cleavage products. Add a cap and a poly(A) tail to pre-mRNA

6. (3) Modification of certain nucleotides on either the base or the sugar moiety. –Add a methyl group to 2’-OH of ribose in mRNA (A) and rRNA –Extensive changes of bases in tRNA

7. RNPs Ribonucleoproteins = RNA protein complexs  The RNA molecules in cells usually exist complexed with proteins  specific proteins attach to specific RNAs  Ribosomes are the largest and most complex RNPs

8. 3-D structure

9. Digital cryo-electron micrography RNP

10. Ribosomes  Protein biosynthetic machinery  Made of 2 subunits (bacterial 30S and 50S, Eukaryotes 40S and 60S)  Intact ribosome referred to as 70S ribosome in Prokaryotes and 80S ribosome in Eukaryotes  In bacteria, 20,000 ribosomes per cell, 25% of cell's mass.  Mass of ribosomes is roughly 2/3 RNA

11. Prokaryotic Ribosome Structure

12. Eukaryotic Ribosome Structure  larger and more complex than prokaryotic ribosomes, but with similar structural and functional properties

13. tRNA PROCESSING, RN ASE P AND RIBOZYMES  tRNA processing in prokaryotes tRNA processing in prokaryotes  tRNA processing in eukaryotes tRNA processing in eukaryotes  RNase P RNase P  Ribozymes Ribozymes

14. tRNA 3-D structure

15. t RNA processing in prokaryotes Mature tRNAs are generated by processing longer pre-tRNA transcripts, which involves 1. specific exo- and endonucleolytic cleavage by RNases D, E, F and P (general) followed by 2. base modifications which are unique to each particular tRNA type.

16. t RNA processing in prokaryotes Primary transcripts RNase D,E,F and P tRNA with mature ends Base modifications mature tRNAs

17. t RNA processing in eukaryotes  The pre-tRNA is synthesized with a 1. 16 nt 5 ’ -leader, 2. a 14 nt intron and 3. two extra 3 ’ -nucleotides.

19. t RNA processing in eukaryotes 1. Primary transcripts forms secondary structures recognized by endonucleases 2. 5 ’ leader and 3 ’ extra nucleotide removal 3. tRNA nucleotidyl transferase adds 5’-CCA-3’ to the 3’-end to generate the mature 3’-end 4. Intron removal

20. RNase P  Ribonuclease P (RNase P) is an enzyme involved in tRNA processing that removes the 5' leader sequences from tRNA precursors

21. RNase P  RNase P enzymes are found in both prokaryotes and eukaryotes, being located in the nucleus of the latter where they are therefore small nuclear RNPs (snRNPs)

22. RNase P  RNA component can catalyze pre-tRNA in vitro in the absence of protein. Thus RNase P RNA is a catalytic RNA, or ribozyme.

23. Ribozyme  Ribozymes are RNAs with catalytic activity that can catalyze particular biochemical reactions depending on their capacity to assume particular structures  RNase P RNA is a ribozyme.  Ribozymes function during  protein synthesis,  in RNA processing reactions, and  in the regulation of gene expression

24. Ribozyme  Self-splicing introns: the intervening RNA that catalyze the splicing of themselves from their precursor RNA, and the joining of the exon sequences

25. Ribozyme  Self-cleaving RNA encoded by viral genome to resolve the concatameric molecules of the viral genomic RNA produced. These molecules are able to fold up in such a way as to selfcleave themselves into monomeric.

26. Ribozyme Ribozymes can be used as therapeutic agents in 1. correcting mutant mRNA in human cells 2. inhibiting unwanted gene expression  Kill cancer cells  Prevent virus replication

27. The Power of RNA interference LOSS OF FUNCTION Easy in yeast Difficult in mammals RNA Interference (RNAi) is able to block selective mRNA

28. RNAi Pathway RNAi = RNA interference siRNA = small interfering RNA siRNP = small interfering Ribonucleoprotein RISC = RNA Induced Silencing Complex Dicer

29. Inhibition the replication of SARS virus by RNA interference

30. mRNA PROCESSING, hnRNPs AND snRNPs  Processing of mRNA Processing of mRNA  hnRNP hnRNP  snRNP particles snRNP particles  5 ’ Capping 5 ’ Capping  3 ’ Cleavage and polyadenylation 3 ’ Cleavage and polyadenylation  Splicing Splicing  Pre-mRNA methylation Pre-mRNA methylation

31. Processing of mRNA  There is essentially no processing of prokaryotic mRNA, it can start to be translated before it has finished being transcribed.  Prokaryotic mRNA is degraded rapidly from the 5 ’ end

32. Processing of mRNA in eukaryotes  In eukaryotes, mRNA is synthesized by RNA Pol II as longer precursors (pre-mRNA), the population of different RNA Pol II transcripts are called heterogeneous nuclear RNA (hnRNA).  Among hnRNA, those processed to give mature mRNAs are called pre- mRNAs

33.  Pre-mRNA molecules are processed to mature mRNAs by 5 ’ -capping, 3 ’ - cleavage and polyadenylation, splicing and methylation. Processing of mRNA in eukaryotes

34. Eukaryotic mRNA processing: overview

35. hnRNP: hnRNA + proteins  The hnRNA synthesized by RNA Pol II is mainly pre-mRNA and rapidly becomes covered in proteins to form heterogeneous nuclear ribonucleoprotein(hnRNP)  The hnRNP proteins are though to help keep the hnRNA in a single- stranded form and to assist in the various RNA processing reactions

36. snRNP particles: snRNA + proteins 1. snRNAs are rich in the base uracil, which complex with specific proteins to form snRNPs. 2. The most abundant snRNP are involved in pre-mRNA splicing, U1,U2,U4,U5 and U6. 3. A large number of snRNP define methylation sites in pre-rRNA.

37. snRNP particles  They are synthesized in the nucleus by RNA Pol II and have a normal 5 ’ -cap.  They are exported to the cytoplasm where they associate with the common core proteins and with other specific proteins.  Their 5 ’ -cap gains two methyl groups and they are then imported back into the nucleus where they function in splicing.

39. Splicing  Introns: non-coding sequences  Exons: coding sequences  RNA splicing: removal of introns and joining of exons  Splicing mechanism must be precise to maintain open reading frame  Catalyzed by spliceosome (RNA + protein)

40. 5’ Capping  Very soon after RNA Pol II starts making a transcript, and before the RNA chain is more than 20 -30 nt long, the 5 ’ -end is chemically modified.  7-methylguanosine is covalently to the 5´ end of pre-mRNA.  Linked 5´  5´  Occurs shortly after initiation

41. 7-methylguanosine (m 7 G)

43. Function of 5´ cap  Protection from degradation  Increasing translational efficiency  Transport to cytoplasm  Splicing of first exon

44. 3’ Cleavage and polyadenylation  In most pre-mRNAs, the mature 3 ’ -end of the molecule is generated by cleavage followed by the addition of a run, or tail, of A residues which is called the poly(A) tail.

45. 3’ Cleavage and polyadenylation  RNA polymerase II does not usually terminate at distinct site  Pre-mRNA is cleaved ~20 nucleotides downstream of polyadenylation signal (AAUAAA)  ~250 AMPs are then added to the 3´ end  Almost all mRNAs have poly(A) tail

46. Function of poly(A) tail  Increasing mRNA stability  Increasing translational efficiency  Splicing of last intron

47. Splicing  the process of cutting the pre-mRNA to remove the introns and joining together of the exons is called splicing.  it takes place in the nucleus before the mature mRNA can be exported to the cytoplasm.

48. Splicing  Splicing requires a set of specific sequences to be present. The 5’-end of almost all introns has the sequence 5’-GU-3’ and the 3’-end is usually 5’-AG-3’. The AG at the 3’- end is preceded by a pyrimidine-rich sequence called the polypyrimidine tract

49.  Sequences of (a) a typical polyadenylation site and (b) the splice site consensus.

50. Spliceosome  Catalyzes pre-mRNA splicing in nucleus  Composed of five small nuclear RNAs (snRNAs) and associated proteins (snRNPs) assembled on the pre- mRNA  Splicing reaction is catalyzed by RNA

52. Splicing cycle

53. Splicing  Introns: non-coding sequences  Exons: coding sequences  RNA splicing: removal of introns and joining of exons  Splicing mechanism must be precise to maintain open reading frame  Catalyzed by spliceosome (RNA + protein)

54. Pre-mRNA methylation  The final modification or processing event that many pre-mRNAs undergo is specific methylation of certain bases.  The methylations seem to be largely conserved in the mature mRNA.

55. ALTERNATIVE mRNA PROCESSING  Alternative processing Alternative processing  Alternative poly(A) sites Alternative poly(A) sites  Alternative splicing Alternative splicing  RNA editing RNA editing

56. Alternative processing  Alternative mRNA processing is the conversion of pre-mRNA species into more than one type of mature mRNA.  Types of alternative RNA processing include alternative (or differential) splicing and alternative (or differential) poly(A) processing.

57. Alternative poly(A) sites  Some pre-mRNAs contain more than one poly(A) site and these may be used under different circumstances to generate different mature mRNAs.  In one cell the stronger poly(A) site is used by default, but in other cell a factor may prevent stronger site from being used.

58. Alternative splicing  The generation of different mature mRNAs from a particular type of gene transcript can occur by varying the use of 5’- and 3’- splice sites in four ways: (i) By using different promoters (ii) By using different poly(A) sites (iii) By retaining certain introns (iv) By retaining or removing certain exons

59. Alternative splicing 1. By using different promoters.2. By using different poly(A) sites.3 By retaining certain introns.4. By retaining or removing certain exons

60.  Sex in Drosophila is largely determined by alternative splicing

61. Alternative splicing the potential for an increase in phenotypic diversity without increasing the overall number of genes. Is achieved by altering the pattern of exons that are spliced together, different proteins can arise from the processed mRNA from a single gene.

62. Alternative splicing Alternative splicing can occur either at specific developmental stages or in different cell types. the calcitonin gene yields an mRNA that synthesizes calcitonin (thyroid) or calcitonin gene– related peptide (CGRP, brain): 2 proteins with distinctly different functions. the α-tropomyosin mRNA have at least 8 different alternatively spliced α-tropomyosin mRNAs.

64. Alternative splicing Many defects in the β-globin genes are known to exist leading to β-thalassemias. Some of these defects are caused by mutations in the sequences of the mRNA required for intron recognition and, therefore, result in abnormal processing of the β-globin primary transcript.

65. RNA editing  AN unusual form of RNA processing in which the sequence of the primary transcript is altered is called RNA editing.  Changing RNA sequence (after transcription)  Two types Base modification (A or C deamination) Base (U) insertion and deletion

67. End Of The lecture Thanks!