HC11 Long and small non-coding RNA

Question 1

lncRNA: involved in

Answer 1

gene regulation, genomic imprinting, X-chromosome inactivation

Question 2

miRNAs: involved in

Answer 2

fine-tuning of gene expression.
Developmental regultion of gene expression

Question 3

si/piRNAs: role in

Answer 3

genome stability.
siRNA: defense against pathogens

Question 4

active enhancer produce:

Answer 4

lncRNAs. also called enhancer RNAs (eRNA)/

Question 5

three categories lncRNA

Answer 5

1. non-functional lncRNA as a result from transcriptional noise
2. non-functional lncRNA of which the transcription process itself is sufficient for their function
3. functional lncRNAs: can act in cis/trans. HOTAIR, Xist, Tsix

Question 6

lncRNA + transcriptial interference

Answer 6

• lncRNA is encoded opposite a protein coding gene. Both genes get transcribed –> two RNA polymerases move in opposite directs –> collide –> transcription in antisense direction (of lncRNA) interferes with transcription of protein coding gene

This way transcriptional repression

Question 7

3 models of lncRNA affecting gene expression

Answer 7

1. guide model:
   - lncRNA bind/recruit transcriptional activator/repressor –> conformational change –> binding of proteincomplex to DNA
2. Decoy model:
   lncRNAs bind to a transcriptional activator/repressor –> conformational change –>
   release of the protein (complex)
3. Scaffold model:
   by binding: lncRNA brings multiple proteins together –> exert certain function

Question 8

Example of lncRNA acting as decoy

Answer 8

GAS5 (lncRNA) –> mimics GRE –> binds to GR –> no transcriptional activation of target gene.

Question 9

Example of lncRNA acting as scaffold

Answer 9

HOTAIR binds to PRC2 and to H3K4me2/3 demethylase –> addition of H3K27me + removal of H3K4me –> repression of multiple target genes.

HOTAIR acts in trans

Question 10

lncRNA and X-inactivation

Answer 10

• Xist has scaffold function (with PcG, moves over the to be inactive X-chromosome)
• Tsix RNA is antisense to Xist –> prevents accumulation of Xist on the to be active X chromosome.

Question 11

ncRNA-a7

Answer 11

lncRNA. Functions as a scaffold for assembly of TF complexes

Question 12

HOTTIP

Answer 12

is a lncRNA: guides H3K4methyltransferases to TSS

Question 13

RNA interference

Answer 13

silencing by small RNAs.
dsRNA is primary trigger of RNAi.

Question 14

general RNA pathway

Answer 14

• Dicer cleaves long dsRNA into smaller fragments (20-30 bp) –> these fragments associate with ARGONAUTE proteins in the RISC complex –> RNAs get unwound –> 1 strand (antisense/guide strand) remains bound to RISC complex –> RISC binds to target RNA and cleaves it.

Question 15

sources of dsRNA

Answer 15

• sequence in inverted orientation –> results in hairpin RNA = dsRNA.
• viral RNA
• sense and antisense RNA production from same sequence
  -TEs
• externally supplied dsRNA
• RNA-dependent RNA polymerase activity

Question 16

results from RNA pathways

Answer 16

• Transcriptional gene silencing: no/less RNA is made. pi/siRNA.
• post-transcriptional gene silencing: RNA is degraded or not translated. mi/pi/siRNA.

Question 17

miRNA pathway

Answer 17

endogenous gene is transcribed –> resulting RNA transcript folds into hairpin structure = pri-miRNA –> Drosha cleaves 5’cap + polyA tail = pre-miRNA –> transported out of nucleus by Exportin 5 –> Dicer cleaves pre-miRNA = miRNA –> miRNA is loaded onto ARGONAUTE –> active RISC complex with the antisense strand attached –> binding to target RNA –> RNA degradation/translation inhibition

Question 18

miRNA + target RNA: degree of matching?

Answer 18

mismatches: translation inhibition
matches: so perfect complementarily: RNA degradation

Question 19

mirtrons

Answer 19

miRNA encoded in introns. this pathway is slighly different: no drosha but first splicing.

Question 20

piRNA pathway

• required for?

Answer 20

piRNA clusters get transcribed –> processing of piRNA –> loading into PIWI proteins –> TE sequences also get described –> transport out of nucleus –> piRNA binds to TE RNA –> cleaving and proccesing of TE.

ping-pong cycle: since every secondary piRNA can recognise new TE/piRNA sequences.

required for: germline development.

PTGS

Question 21

piRNA return to nucleus sometimes: effects

Answer 21

mediate H3K9me of TEs –> TGS

Question 22

siRNA pathway

Answer 22

PTGS

dsRNA –> cleaved by Dicer –> loaded onto RISC –> one strand remains bound to RISC –> RISC activation –> target binding –> target RNA cleavage.

have antiviral role. in mammals its limited to ESCs/early development. Can be used to develop drugs.

Question 23

Knocking down lncRNAs: how?

Answer 23

using small RNAs in a vector
- tissue specific promotor sequence
- inverted sequences
- in between the inverted sequences: spacer
- terminator