miRNAs

Question 1

How is mature microRNA formed?

Answer 1

1. Transcription of pri-mRNA.
2. Processed by Drosha to form and DGCR8 to form pre-mRNA.
3. Pre mRNA is transported from the nucleus to cytoplasm by exportin 5 protein.
4. Second cleavage by DICER which cuts the hairpin loop generating miRNA duplex.
5. Loaded onto argonaute 1-4 to form RNA-induced silencing complex RISC.
6. Translational inhibition and deadenylation can occur of mRNA.

Question 2

How is mature endo-siRNA formed?

Answer 2

1. The formation of dsRNA precursor from endogenous sources:inverted repeats, transposons.
2. Processing by Dicer into siRNA duplexes.
3. Strand selection to determine the guide strand.
4. Loading onto Argonaute proteins to form RISC.
5. Target mRNA recognistion and gene silencing by either cleavage or translational repression.

Question 3

Where does DROSHA cleave?

Answer 3

5’ and 3’ ends. Form hairpin primary precursor.

Question 4

Which is more important: the canonical pathway or non canonical?

Answer 4

Neither. Both just use different enzymes.

Question 5

What does AGO2 do?

Answer 5

Supports Dicer by cleaving the 3’arm of some pre-miRNAs.

Question 6

What does GW182 do?

Answer 6

Core component of the miRISC complex and an important part of the processing/degradation of mRNAs.

Question 7

What are P-bodies and stress granules?

Answer 7

For mRNA regulation. Repressed mRNAs can accumulate in discrete cytoplasmic foci: Processing bodies (P-bodies), used for the storage and decay of miRNAs. Enriched in proteins for translational repression, mRNA deadenylation, decapping and degradation.

Question 8

What is the seed region?

Answer 8

The primary specificity determinant. A limited region of sequence at the 5’ end of miRNA. How most miRNAs bind to their target. Contributes most of the energy required for target binding.

Question 9

What is the seed sequence?

Answer 9

First 2-8 nucleotides starting at the 5’ end of the miRNA. A seed match is a Watson-Crick match between an miRNA and its target.

Question 10

What are the several types of seed matches?

Answer 10

6mer: Perfect WC match for miRNA seed and mRNA for 6ntds. 7mer-m8: A perfect match from nucleotides 2-7 of miRNA seed. 7mer-A1 same but in addition to an A across from the miRNA nucleotide 1. 8mer: A perfect WC match from nucleotides 2-8 and an A across from the miRNA nucleotide 1.

Question 11

What are the common features of miRNA-mRNA to predict miRNAs targets?

Answer 11

Seed match, conservation, free energy, site accessibility. If seed region is conserved suggests an important target.

Question 12

Can miRNAs target multiple mRNAs?

Answer 12

Yes if the seed region is the same.

Question 13

How have cluster miRNAs arisen?

Answer 13

Polycistronic clusters from which several miRNAs can be co-expressed. Evolution of these came from gene duplications. Frequently part of seed families.

Question 14

What are the three mechanisms of miRNA mediated gene silencing?

Answer 14

Repression of translation initiation by preventing ribosome subunits from binding. Post-initiation inhibition as the loading complex makes the ribosome drop off. Target mRNA destabilization. Most drastic output. Exonuclease causes deadenylation.

Question 15

How could miRNAs be therapeutic options?

Answer 15

1. miRNA sponges. Bind to the miRNA to prevent it from binding to the target mRNA.
2. Anti-miRNAs are complementary so compete with the mRNA for binding to the miRNA.
3. Small molecules bind to the structure that could potentially be pathological.

Question 16

What are the risks of miRNA therapies?

Answer 16

Capacity for binding to multiple targets increases off-target effects. Highly regulated spatiotemporal expression complicates development.

Question 17

How do you improve specificity?

Answer 17

With target protectors. miRNA target site protectors are antisense oligonucleotides that bind to microRNA target site of an mRNA preventing microRNAs from gaining access to that site.

Question 18

What is the function of piwi interacting RNAs (pi-RNAs)?

Answer 18

Defense of germline genome from transposon mobilization. Also post transcriptional gene regulation in the cytoplasm.

Question 19

How are piRNP complexes formed?

Answer 19

1. Transcription of piRNA precursors from clusters in the genome.
   
   2. Primary processing and cleavage by Zucchini to define the 5’ end.
   3. Trimming and methylation at the 3’ end.
   4. Loading onto PIWI proteins to form piRISC.
      Target recognition and the ping-pong cycle for amplification and transposon silencing.