Class 13 Slides: small RNAs in gene regulation

The generation of different cell types depends on

gene regulation

Gene regulation

controls cell identity by selectively turning genes “on” or “off” in different cell types

genome in cells

Cells with the same genome, but different sets of genes “on” that determine the sets of proteins important for that particular cell type: Keratin produced in skin cells, Myosin produced in muscle cells and so on

Multiple layers of regulation

fine-tune gene expression

Small interfering RNAs (siRNAs), origin, what do they do

can have endogenous or exogenous origin

Responsible for mRNA cleavage and heterochromatin formation

MicroRNAs (miRNAs), origin, what do they do

represent a new superfamily of genes: endogenous origin

Mechanism: mRNAs degradation, translation repression

The first microRNA was discovered in

C. elegans

The 22nt lin-4 miRNA regulates lin-14 via sites in

its 3’UTR

Temporal expression of

lin-4 miRNA downregulates LIN-14 protein expression

Discovery of second miRNA

let-7 prompted future work on miRNAs

miRNAs have been characterized

across the animal kingdom, with many highly conserved across diverse species

Overview of miRNA biogenesis

translation inhibition, mRNA degradation

primary miRNA transcript capped, polyadenylated, up to 10kb in length

precursor microRNA hairpin (~70nt)

• Imperfect miRNA-mRNA base pairing is allowed.

• G::U base-pairing is allowed.

• Short seed sequence is the initial “anchor” for miRNA-mRNA pairing.

• Nucleotides 2–7 or 2–8 from the 5′ end of the miRNA, essential for target recognition and binding

miRNA 1

precursor microRNA hairpin (~70nt)

miRNA base pairing

• Imperfect miRNA-mRNA base pairing is allowed.

• G::U base-pairing is allowed.

miRNA vital sequence

Short seed sequence is the initial “anchor” for miRNA-mRNA pairing.

• Nucleotides 2–7 or 2–8 from the 5′ end of the miRNA, essential for target recognition and binding

miRISC cause

mRNA degradation by removal of the polyA tail and 5’ cap, degradation by nucleases

This is the main way that miRNAs control gene expression

• Translation inhibition is less common

miRISC mRNA degredation step 1

1. Recruitment of the CCR4-NOT1 Deadenylase complex (removes poly A tail)

miRISC mRNA degredation step 2

Recruitment of 3’-5’ exonucleases recognize deadenylated mRNAs as bad and chew them up

miRISC mRNA degredation step 3

Recruitment of Decapping complex removes 5’ cap complex

miRISC mRNA degredation step 4

Recruitment of 5’-3’ exonucleases recognize loss of 5’ cap of mRNA as bad and chews them up

Endogenous siRNAs are encoded

in the genome

transcription of a single RNA that can base pair with itself

transcription of two overlapping RNAs that base pair with each other, forming double-stranded RNA

siRNAs processing location

in the cytoplasm

siRNAs processing step 1

1. Processing DICER

siRNAs processing step 2

2. Loading siRISC /miRISC

siRNAs processing step 3

Passenger strand ejected

siRNAs processing step 4

Target binding

siRNAs processing step 5

Target cleavage by Ago in coding region RNA interference

dsRNA induces

potent silencing (interference)

some dsRNAs are a perfect reverse-complement of the target sequence

RNAi has been used to

investigate gene functions

RNAi screens allow

phenotypic observation of knock-down of any gene

Create a collection of dsRNAs to target (nearly) every gene in the genome

Add one dsRNA to each well of a tissue culture dish

Observe “knockdown” phenotypes

The centromere encodes

siRNAs in fission yeast

• Centromeres have a structural role in chromosome segregation in mitosis and meiosis

• If transcribed, dh transcripts form dsRNA which is processed into siRNA

• siRNA binds a nuclear version of RISC

• Cleaves centromere-derived transcripts in the nucleus

• Directs heterochromatin at centromere

Nuclear RISC found in C. elegans