W7L2 mechanism of siRNA and miRNA action

Question 1

What is RISC

Answer 1

The effector complex for gene silencing

Question 2

Formation of RISC

Answer 2

miRNA/siRNA duplex-Dicer complex present in the cytoplasm
-miR:miR* duplex/siRNA duplex associated with Dicer
-R2D2 recruited to Dicer –RLC formed with R2D2 binds to the end of the duplex with the highest thermodynamic stability
-miR:miR* duplex/siRNA duplex loaded into Ago
-A cytoplasmic RISC-loading complex (RLC) is formed and the duplex is loaded into AGO, AGP and associated protein form a cytoplasmic multi-component ribonucleoprotein complex – RNA-induced silencing complex (RISC)
-the duplex is then unwound and one strand is ejected, miRNA is always ejected . The idendity of the passenger strand depend on R2D2 orientation

Question 3

RISC idendity

Answer 3

Active RISC has one strand of the duplex (miRISC/siRISC)
Gene silencing involves base-pairing between miR/guide RNA in RISC and target transcript

Question 4

Some RISCs slice their targets

Answer 4

Extensive pairing between RISC and target transcript
- near perfect pairing to sequences located in any region of the transcript
* Gene silencing associated with endonucleolytic cleavage (‘slicing’) of target mRNA
* Slicing of target mRNA occurs within the target recognition site
* Cleaved mRNA is subsequently degraded
-cleavage induced degradation applies to siRNA and plant miRISC

Question 5

RISCs that do not slice their target

Answer 5

-limited paring between RISC and the target transcript at the 3’ UTR (pairing of multiple short repetitive sequences)
* Number/efficiency of miRNA binding sites in 3’ UTR determines extent of repression
* Gene silencing reduces protein synthesis or causes mRNA destabilization (deadenylation and decay)
-Block in protein synthesis and deadenylation applies to animal miRISCs

Question 6

Mechanism used by RISC to block protein synthesis

Answer 6

Animal miRISCs associate with the target mRNA and recruits PABPC-interacting protein and Deadenylase complex
-this lead to Shortens poly(A) tail/decapping/degradation
Deadenylase complex also recruits factors that block recruitment of the 43S PIC

Question 7

Rules for Predicting targets of plant miRNAs

Answer 7

-Near perfect complimentary revels some rules on target identification
5’ region (2-12) strict complementarity – only 1 mismatch tolerated
No mismatch in cleavage site (10-11)
3’ region (13-21) can tolerate up to 4 mismatches, but not more than 2 in a row

Question 8

Target recognition in plants approach

Answer 8

Approach – Scan genome sequence for regions with 4 or less mismatches to a particular miRNAs

Question 9

Observation from target recognition in plants

Answer 9

1. Target one gene/closely related members of a gene family
2. miRNA target site is typically located in the coding sequence of targeted transcript
3. Mainly transcription factors involved in developmental patterning, cell differentiation and nutrient homeostasis
   Regulatory potential of plant miRNAs is limited to one or more members of a single gene family

Question 10

rules for Predicting targets of animal miRNAs

Answer 10

Typical miRNA recognition site in 3’ UTR – based on the analysis of lin-4 and let-7 targets
Rules:
Strict complementarity over 7nt of seed region (position 2-8)
Maybe binding at position 1/9
Central bulge between position 10-12
Moderate complementarity position 13-16

Question 11

Target recognition in animals Approach

Answer 11

§ Search for 3’ UTRs that have ≥2 seed match sites
§ Use genomic sequence to identify related genes and align 3’ UTRs
§ Search within aligned 3’ UTRs for occurrence of conserved seed match sites

Question 12

General observation for target recognition in animal

Answer 12

1. One miRNA can have many unrelated targets
2. One gene can be targeted by multiple miRNAs with different seeds
3. Targets are enriched for transcription factors involved in cell proliferation, cell differentiation, physiological pathways, and apoptosis
   -Regulatory potential of animal miRNAs is massive

Question 13

miRNA display pattern and role

Answer 13

-miRNA expression is remarkably specific and diverse
Detected during embryonic and postnatal development and in adult tissues
– miRNAs have roles in differentiation and maintenance of tissue identity

Question 14

types Regulatory roles of miRNA

Answer 14

-Switch – complete suppression of gene activity (temporal or spatial)
-Insulator – limits gene activity. Reinforces transcriptional regulation
Tuner – limits gene activity
control levels of gene activity within cells/tissues

Question 15

miRNAs that function as switches

Answer 15

Question 16

Example of a spatial switch miRNA

Answer 16

Blood development in zebrafish – use RNA in situ hybridization to look at the expression pattern of miR451 and target gene gata2
-miRNA only express in antertoir ICM, not posterior.
miRNA activity rapidly clears target gene activity from cells/tissue

Question 17

Consequences for loss of a spatial switch

Answer 17

Question 18

miRNAs that function as insulators

Answer 18

Question 19

Example of an insulator miRNA

Answer 19

Question 20

Consequences for a loss of function for an insulator

Answer 20

Question 21

miRNAs that function as tuners

Answer 21

Question 22

Example of a tuner miRNA

Answer 22

Question 23

Consequences for loss of a tuner miRNA

Answer 23

Question 24

Key info in miRNA activity

Answer 24

4. Many miRNAs display precise spatiotemporal expression patterns
5. Majority of animal miRNAs sculpt the transcriptome (insulators/tuners)
6. Majority of plant miRNAs act as developmental switches

Question 25

Observation from target recognition in plants