Eukaryotic regulatory RNAs

Question 1

Regulatory RNAs

Answer 1

Base pair with RNA/DNA targets. Binding can block proteins, change RNA structure and tether a protein

Small interfering RNA (siRNA)
Micro RNA (miRNA)
Piwi-interacting (piRNA)
long non-coding (lncRNA)

Question 2

miRNA processing

Answer 2

Primary transcripts (pri-miRNAs) are capped, polyadenylated and may contain introns
Folds into a stem loop structure
5' and 3' extensions removed by microprocessor complex (Drosha and DGCR8) to produce a hairpin (pre-miRNA).
Hairpin exported from nucleus. PAZ domain of Dicer binds the 3' end and cleaves pre-miRNA to give two miRNA strands

Question 3

siRNA processing

Answer 3

Derived from dsRNA from endogenous or exogenous sources.
No Drosha dependent reaction and no cleavage in the nucleus
Dicer chops up long dsRNA by sequential cleavage to give siRNA:siRNA molecules

Question 4

Argonaute proteins

Answer 4

Four domains N, PAZ (binds 3’ RNA end), Mid (5’ RNA end) and PIWI (interacts with whole RNA)

PAZ, Mid and PIWI orient bound RNA to facilitate scanning of cellular molecules

Question 5

sRNA loading on to Argonaute

Answer 5

One strand (mi/siRNA) retained and other is removed (sorting).
Retained guide strand involved in silencing target RNA
Guide RNA-Argonaute complex is called RISC

Question 6

Gene silencing by miRNA

Answer 6

miRISC pairs target RNA via 2-8 nucleotide seed sequence. 3’ end of miRNA tightly bound by PAZ domain.
miRNA binding sites found in 3’ region of target and can be targeted by several miRISCs
Pairing is imperfect
No cleavage but causes translational repression

Question 7

Gene silencing by siRNA

Answer 7

siRISC can bind along its full length.
This releases PAZ domain and induces a conformational change, activating PIWI domain
PIWI domain cleaves target RNA

Question 8

C. elegans miRNA

Answer 8

lin4 gene encodes miRNA that binds 3’ UTR of lin14 mRNA preventing translation

Question 9

piRNA processing

Answer 9

piRNAs generated from long transcripts from specific loci (no dicer)
Precursor transcript loaded onto a PIWI protein.
PAZ domain at N terminus provides a binding pocket for the 3’ end of the RNA
5’ phosphate anchored by MID domain
RNA cleavage occurs in the PIWI domain

Question 10

piRNA functions

Answer 10

Transposon silencing defends the germline expression
Antiviral defence in somatic tissue
Regulation of gene expression.

Question 11

rasiRNAs

Answer 11

repeat associated small interfering RNAs
cleave target RNA
Epigenetic effects through association with chromatin to induce methylation (repression)

Question 12

lncRNAs

Answer 12

Transcription SRG1 lncRNA through SER3 promoter blocks initiation of SER3 transcription in yeast

Transcription of lncRNAs upstream of fbp1 opens up chromatin

Question 13

MiRNA and B cell development

Answer 13

Knockout of dicer arrested pro-B to pre-B transition