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RNA PROCESSING AND RNPs

RNA PROCESSING AND RNPs. 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

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RNA PROCESSING AND RNPs

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

  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 AAAAAA

  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. tRNAPROCESSING, RNASEPANDRIBOZYMES • tRNA processing in prokaryotes • tRNA processing in eukaryotes • RNase P • Ribozymes

  14. tRNA 3-D structure

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

  16. tRNA processingin prokaryotes Primary transcripts RNase D,E,F and P tRNA with mature ends Base modifications mature tRNAs

  17. tRNA processingin eukaryotes • The pre-tRNA is synthesized with a • 16 nt 5’-leader, • a 14 nt intron and • two extra 3’-nucleotides.

  18. tRNA processingin eukaryotes • Primary transcripts forms secondary structures recognized by endonucleases • 5’ leader and 3’ extra nucleotide removal • tRNA nucleotidyl transferase adds 5’-CCA-3’ to the 3’-end to generate the mature 3’-end • Intron removal

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

  20. 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)

  21. 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.

  22. 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

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

  24. 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.

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

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

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

  28. Inhibition the replication of SARS virus by RNA interference

  29. mRNAPROCESSING, hnRNPsANDsnRNPs • Processing of mRNA • hnRNP • snRNP particles • 5’Capping • 3’Cleavage and polyadenylation • Splicing • Pre-mRNA methylation

  30. 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

  31. 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

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

  33. Eukaryotic mRNA processing: overview

  34. 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

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

  36. 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.

  37. 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)

  38. 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

  39. 7-methylguanosine (m7G)

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

  41. 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.

  42. 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

  43. Function of poly(A) tail • Increasing mRNA stability • Increasing translational efficiency • Splicing of last intron AAAAAA

  44. 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.

  45. 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

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

  47. 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

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