RNA biology lecture 3

Question 1

pre-mRNA splicing overview

Answer 1

• In more than 95% of genes, involves transcription of exon/intron structure, resolved w/ splicing
• Coding info = arranged in exons separated by introns
• Need to remove introns

Question 2

Removal of introns

Answer 2

• Splicing = mRNA-mediated trans-esterification → 2 sequential breakages = rejoining of sugar phosphate backbone
• Mediated by SNURPs in spliceosome
• Adenosine in intron carries nucleophilic attack w/ 2’OH onto phosph of 1st nuc in exon
• Exon has free 3’OH attacks exon/intron border → intron released as Lariat

Question 3

Protein encoding genes vary in size

Answer 3

• B-globin gene = 146aa, 1.6kb
• Titin = 34,350 aa, 283 Kb
• Dystrophin = 3,685 aa but 2.4Kb (30,770 introns)

Question 4

Cis-elements in pre-mRNA

Answer 4

• Specific sequences surrounding exon/intron structure at start or end (5’SS or 3’SS gives directionality)
• 5’SS = characterised by sequences in exon + intron, around 10 conserved nucleotides
• 3’SS = us 3 nucleotides of splice site, pyrimidine trap followed by branch point

Question 5

Spliceosome

Answer 5

• Has over 200 proteins, 5 RNA players: U1,2,4,5,6snRNA
• snRNA = SM bs, 200 nt in length, conserved 2o structure, have ds region of RNA, ss region e.g. 5’ end, important to recognise specific 5’/3’ sequences
• U4+6 interact together via 2p structures → catalytic centre
• 2 types of protein the snRNAs associate w/ : RNA specific e.g. 70K vs common e.g. SM proteins

Question 6

Assembly of spliceosome on pre-mRNA

Answer 6

• 3’ + 5’ SS = recognised by biding of SF2
• U1 snRNA recognises 5’ SS by GU at starts of most introns
• 5’ part of U1 = ssRNA + bp w/ exon/intron
• U2AF recognises pyrimidine track + AG at 3’ SS
• U2 snRNA helps other proteins assoc. w/ branch point, A at branch point = bulged out
• U4,5,6 snRNA appear as a 3, U4+6 join
• Causes ↑ rearrangement, U1 + 4 ejected
• U6 snRNA replaces U1 at 5’SS, U6 + 2 interact
• Complex RNA-RNA interaction, 2’OH + exon 1 brought close
• 2’OH carries out nucleophilic attack on exon-intron border
• After 1st translocation, have 2nd major 2nd rearragement → 2nd transest.
• 3’ SS executes 2nd nuc attack

Question 7

Co-transcriptional splicing

Answer 7

• Splicing = post-translational in complex w/ RNA Pol II(phosph at Ser5)
• Free 5’SS remains assoc w/ complex until polymerase transcribes sequences ds of exon
• Co-transcription = important

Question 8

Exon junction complex

Answer 8

• Splicing also marks mRNA
• Co-immunoprecip shows EJC assoc. w/ spliced mRNA
• Proteins deposited 20-40 us of exon-intron junction
• In cytoplasm, some factors release, others join

Question 9

Catalysis of RNA splicing

Answer 9

• Discovery of self-splicing RNA = importance of snRNA
• Self splicing introns = group I/II, fold + self-cleave, similar reaction to spliceosome but w/o protein
• Suggests some catalytic activity in residues in RNA not protein
• E.g. group II RNA
• rRNA have introns that need to be excised, intronic regions fold into structures, 2’OH carries out nuc. attack, leaves 3’OH w/ 2’5’ linkage of intron, 3’OH nuc. attack on intron border → 2 exons fuse

Question 10

Circular RNAs

Answer 10

• Involved in gene regulation + disease
• Results from backspacing (ds 5’SS attacks 3’SS of previous exon → circular RNA, bp btw repeat sequences in introns, circ RNAs can be exported-
• E.g. = role as sponge for miRNA + RNA binding protein