RNA biology lecture 9

Question 1

Processing of primary ribosomal RNA transcript

Answer 1

• rRNA = 80% of cell RNA mass
• 100s of rDNA genes = tandem arrays on chromosomes 18,14,15
• Regions form nucleolus organiser region
• After transcription by RNA pol 1, no. of nucleotides in Io RNA are mod

Question 2

Processing of rRNA precursors in nucleolus

Answer 2

• rRNA has PolI-specific promoter, recog by TF SL1
• Pol I transcribes genes - pre-ribosome RNA
• Transcription stopped at terminators
• ITS = btw 18S + 5.8S, ITS-2 = btw 5.8S-28S, 3’ETS = after 28S
• Primary transcript = modified → 18, 5.8 + 28S RNA
• 5S RNA transcribe by RNA Pol III in nucleoplasm, imported to nucleolus
• In nucleolus, assemble w/ ribosomal proteins

Question 3

snoRNP cleavage

Answer 3

• pre-rRNA sequences like in ITS-1 = recognised by endoribonuc MRP, releases 20 + 32S ribosomal precursor
• U8 snoRNA recognises ITS-1 + 3’ETS
• tRNA modified by methyl + pseudouridyl, guided by SnoRNA
• Las1 endonuc uses seq in ITS2 to make precursors for 5.8 + 28S rNRA
• 5’-3’ of XRN2 also helps

Question 4

tRNA modification

Answer 4

• SnoRNAs, 2 types
  1. BoxC/D snoRNAs (have BoxC, sequences after = conserved, sequences btw C+D boxes in snoRNA hybridise w/ equivalent sequences on rRNA, identifies nucleotides that can be subject to modification in tRNA

2. BoxH/ACA snoRNAs (contain conserved H box consisting of consensus ANANNA btw 2 stem loops + ACA 3 nuc away from 3’ end)

Question 5

5S rRNA

Answer 5

• Transcribed by RNA Pol III in nucleolus
• Intragenic promoters, recognised by TFIIIA,B+C that recruit RNA Pol III
• Assoc w/ LA protein, protects from degradation
• Ribosomal proteins involved in transfer of 5’ to nucleoplasm
• 5S RNA expression regulated by altered reg. feedback
• Zinc finger of TFIIIA can associate w/ 5S RNA when in surplus

Question 6

tRNA processing in eukaryotes

Answer 6

• Precursor tRNA have extended 5’ + 3’ ends, can have introns
• Trimmed by endonucleases
• Nucleotidyl transferase adds CCA to 3’ end
• Introns spliced by complex of sen proteins, leaves tRNA at 3’ w/ cyclic phosph + 5’OH
• Phosphodiester modifies 3’, kinase modifies 5’OH
• Now express anticodon

Question 7

snRNA/ RNP processing

Answer 7

• snRNPs inc U1, 2, 4 + others U7 that process replication-dependent histone genes
• Most transcribed by RNAPII in nuc then exported using Fox to cyt where assoc w/ SMN
• Mutations in MSN = spinal muscular atrophy
• SMN charge snRNAs w/ SM binding proteins, cap of snRNA added m7G

Question 8

Very small ncRNA

Answer 8

• miRNA = 22nt, assoc w/ 3’UTR
• siRNA = 20-25nt, similar to miRNA
• piRNA = 25-30nt long, assoc w/ PIWI → piwi induced silencing

Question 9

miRNA processing

Answer 9

• Transcribed from Pol II
• pre-miRNA folded into specific structures, recognised by DICER, chop stem loop
• Smaller stem loops exported to cytoplasm where assoc. w/ dicing complex inc Argo1 + Dicer1, process 80 nt pre-miRNA → 28nt miRNA
• One ds ejected, remaining ss-RISC complex assoc w. target mRNA to inhibit mRNA
• siRNA assis w. Argo2/Dicer2 →21nt in siRISC

Question 10

RNAi as a tool

Answer 10

• Exogenous delivery of dsRNA, comp to sequences of mRNA, mRNA knockdown

Question 11

Regulating gene expression by ncRNA

Answer 11

1. snRNA in complex that phosph CTD of RNA pol II
   - U1 snRNA involved in both spliceosome + TFIIH
   - Interaction of U1 snRNA-TFIIH stimulates transcription initiation + re-initiation
   - pTEFb = -vely regulated by 7SK snRNA, inhibit Cdk9
2. B2 RNA inhibits transcription of RNA pol II
   - Species specific
   - Heat shock in mouse cells induces Pol III transcribed B2 RNA synthesis
   - B2 RNA directly assoc w/ RNA pol II + inhibits transcription of NON heat shock genes
   - Expression of heat shock = unaffected
   - B2 assoc w/ RNA pol II, incorp into PIC of non-heatshock gene, prevents CDk7 phosph of CTD → prevents promoter escape