MicroRNAs and RNA interference

Why don’t mRNA and protein levels always correlate?

Because translation efficiency

What are the two main types of gene expression control?

Qualitative control via splicing (produces multiple proteins from one mRNA) and quantitative control via mRNA stability (controls translation amount).

What are the two size-based classes of non-coding RNAs?

Long ncRNAs (>200 bp) that act in the nucleus for transcriptional silencing

What plant experiment led to the discovery of RNA silencing?

Inserting extra copies of the CHS gene in petunias caused white flowers instead of deeper purple due to transgene-induced gene silencing (TIGS).

What did Transgene-Induced Gene Silencing (TIGS) show?

That adding transgenes can silence endogenous genes in trans; the effect can spread and be heritable.

What was the first discovered microRNA (miRNA)?

lin-4 in Caenorhabditis elegans.

What does lin-4 regulate?

It binds to conserved sequences in the 3′ UTR of lin-14 mRNA to repress its expression.

Who discovered RNA interference (RNAi) and when?

Andrew Fire and Craig Mello in 1998 (Nobel Prize 2006).

What molecule triggers RNA interference?

Double-stranded RNA (dsRNA)

What is the result of RNA interference?

Degradation of target mRNA with complementary sequence to the introduced dsRNA.

What are the four main steps in siRNA/miRNA-mediated gene silencing?

1) Drosha processes pri-miRNA into pre-miRNA 2) Dicer cleaves pre-miRNA into 21–23 nt fragments 3) RISC complex incorporates siRNA/miRNA 4) RISC binds target mRNA causing repression or degradation.

What does Drosha do?

It cleaves pri-miRNA into ~70 nt pre-miRNA precursors for Dicer processing.

What does Dicer do?

It cuts dsRNA or pre-miRNA into ~21–23 nt fragments for use in silencing.

What does the RISC complex contain?

Argonaute (Ago) proteins and a single siRNA or miRNA strand.

What happens when RISC binds target mRNA?

The mRNA is destabilized or degraded via deadenylation

How many Argonaute proteins exist in mammals and which has slicing activity?

There are AGO1–4; only AGO2 has endonuclease (slicer) activity.

How does siRNA-mediated silencing differ from miRNA-mediated silencing?

siRNA binds fully complementary sites and causes AGO2-dependent mRNA cleavage; miRNA binds partially complementary sites and represses translation or destabilizes mRNA.

How many miRNAs exist in humans?

Around 1500

How many mRNAs can one miRNA regulate?

Each miRNA can target hundreds of mRNAs

What biological processes are miRNAs involved in?

Development

How is RNAi used to study gene function?

By using siRNA or shRNA libraries to knock down mRNA expression and study loss-of-function effects.

Why must siRNA design be careful?

Short hairpins may have off-target effects by binding to unintended 3′ UTRs.

Why is C. elegans a good model for RNAi research?

It shows highly efficient RNAi

Which C. elegans mutants are hypersensitive to RNAi?

eri-1 and rrf-3 mutants.

Can RNAi spread systemically in C. elegans?

Yes

Summarize the function of small RNAs.

siRNA and miRNA regulate mRNA post-transcriptionally by guiding RISC to target mRNAs for degradation or translational repression.

Which enzymes process small RNAs?

Drosha and Dicer.

What does the RISC complex do?

It uses a guide RNA (siRNA or miRNA) to target complementary mRNAs and silence gene expression.

What is the main difference between siRNA and shRNA?

siRNA is a short duplex RNA used directly