Lecture 37: RNA Interference (RNAi) in Prokaryotes

Andrew Fire and Craig Mello discovered what and when?

• they discovered that the expression of any specific gene could be strongly and reversibly inhibited by introducing a double-stranded RNA containing a base sequence from that gene’s mRNA

• discovery made in 1998

When Fire and Mello injected single-stranded RNA complementary to a particular mRNA in embryos, what happened?

there was a modest reduction in gene expression

When Fire and Mello injected double-stranded RNA into embryos, what happened?

the expression of that gene was effectively eliminated

True or False: RNA interference (RNAi) has been reproduced in many different eukaryotes but not in prokaryotes.

true

What does RNAi result from?

transient destruction of the gene’s RNA and doesn’t damage the gene itself

What does RNAi produce?

a knockdown of gene expression

What can RNAi be used to study?

it can be used to study the function of genes for which a permanent knockout would be lethal

When can RNAi be activated?

when dsRNAs either enter the cell outside or from by base-pairing with the cell’s own RNAs

What is the RNAi process?

1. Dicer cleaves the dsRNA more or less randomly into 22 bp fragments

2. The 22 bp siRNA associates with RISC

3. RISC binds the mature double-stranded siRNA

4. Double-stranded siRNA denatures

5. The guide strand of the siRNA targets RISC to RNAs that contain a complementary sequence

6. The second/passenger strand is usually degraded/inhibited

What are the 22 bp fragments produced by Dicer called?

siRNAs

What levels can RNAi of gene expression occur at?

• the siRNA-RISC complex can bind to a complementary mRNA and prevent translation

• the siRNA-RISC complex can cleave and degrade target mRNA

What does RISC contain?

multiple proteins including Argonuate

What is Argonaute?

an RNAse

What does Argonuate contain?

a PAZ domain

What does Argonaute use its PAZ domain for?

• to recognize the 3’ end of the guide siRNA

• to position the target mRNA so that it can cleaved by Argonaute’s RNAse domain

How can the siRNA-RISC complex be used for amplicfication?

the target mRNA can be used as a template for producing more siRNA molecules

What was RNAi used to do in the arctic apple?

to silence genes, specifically the browning enzymes to reduce food waste

What was RNAi used to do in the innate potato?

• to cultivate potatoes that don’t bruise

• to have reduced ability to produce carcinogen acrylamide when fried

• to have blight resistance

• to contain a reduced sugar amount

What is important to know about the evolutionary history of RNAi?

• The siRNA pathway is found in most eukaryotes and is thought to have originated early in their evolution

• siRNA pathway may have been a defense mechanism against RNA viruses

• Animals and plants took advantage of this ancient molecular pathway by synthesizing their own double-stranded regulatory RNAs known as microRNAs (miRNAs)

Why were siRNAs said to have been a defense mechanism against RNA viruses?

mutations in Dicer/Argonaute led to disrupted resistance to viral infections

How does the central dogma and microRNAs relate?

the discovery of microRNAs revealed that animal and plant genomes synthesize an entire class of gene products whose function is to fine-tune by down regulating the expression of other, protein-coding genes

How many distinct miRNAs does the human genome encode?

more than 5,000 miRNAs

What can mutations in miRNAs cause?

diseases such as cancer

How are miRNAs unlike siRNAs?

miRNAs are encoded by genes and are a functional component of the organism’s own genome

What are miRNA genes transcribed by?

RNA Pol II and the primary transcription

Do miRNA’s undergo post-transcriptional modification?

yes, they are capped and polyadenylated

True or False: Some miRNAs are transcribed from their own unique gene while other miRNAs are located within the introns of protein-coding genes.

true

When are miRNAs that are located within the introns of protein-coding genes process?

after the introns have been spliced out of the pre-mRNA

What does each functional miRNA arise from?

a stem loop in the secondary structure of the primary transcript

What is the miRNA regulatory process?

1. Drosha cleaves the pri-miRNA within the nucleus

2. Pre-miRNA is transported to the cytoplasm

3. Dicer cleaves off the terminal loop of the pre-miRNA

4. PAZ domain recognizes the 3’ overhang of the pre-miRNA

5. Dicer measures about 22 bases from each Drosha cut site and cleaves the phosphodiester bonds at two locations

When is pri-miRNA considered pre-miRNA?

when the miRNA releases the stem loop

How does Drosha determine the cleavage sites?

by the shape of the stem loops, not by sequence

What causes bulges in the stem loops?

imperfect base-pairing

Where does Drosha cleave?

a location at the bulge that separates into upper and lower stems

What does cleavage by Drosha leave?

a 3’ overhang

Are cleavage sites random in miRNA?

no, but they are random in siRNA

True or False: The PAZ domain of Dicer is structurally homologous to the PAZ domain found in Argonaute.

true

What does Dicer leave?

a 3’ overhang that is 2 nucleotides in length

True or False: Both Drosha and Dicer process stem-loops on the basis of their size and shape, not base sequence, so there are essentially no limits on the base sequence of the miRNAs that can be produced.

true

True or False: Both siRNAs and miRNAs associate with RISC.

true

What does the guide strand of the miRNA target and what for?

the guide miRNA directs RISC to cellular RNAs with a complementary sequence to either inhibit translation or cause degradation

True or False: In contrast to siRNA, a cell’s miRNA’s have evolved to have sequences that match specific protein-coding mRNAs; thus, each miRNA will specifically down-regulate the expression of one or a few complementary genes.

true

What does RNAi involve in regards to miRNA?

base-pairing between the miRNA and its target mRNA

In C. elegans, what does lin-4 produce?

a 25-base miRNA molecule

What can lin-4 miRNA base pair with?

any 7 nucleotide sequences in the 3’ UTR of the lin-14 mRNA

What happens when the lin-4 miRNA anneals to the lin-14 mRNA?

expression of lin-14 protein reduces

How was lin-4 identified?

through a genetic screen in C. elegans

What does lin-4 do?

it regulates gene expression during development as part of a clock that induces transitions from stage of development to the next

True or False: For many miRNAs, the guide strand and the passenger strand are the two distinct sides of the stem loop, but for some miRNAs, either strand can be used as the guide strand because the two guide strands have different base sequences—inhibiting different target miRNAs.

true

True or False: Recent work has shown that in some cases, the miRNA : RISC complex can enter the nucleus and silence transcription of the gene from which the target mRNA originates.

true

What may transcription silencing in the nucleus from the miRNA:RISC complex involve?

base-pairing between the miRNA and the pre-mRNA before transcription is complete

In animals, what is the third major class of short regulatory RNAs?

piRNAs

What are piRNAs transcribed from?

specific genes within the animal’s own genome

Where are piRNAs primarily transcribed?

in cells of the germ line

True or False: piRNAs interact with Piwi proteins germ cells.

true

Within germ line cells, what do piRNAs and Piwi proteins do?

act together to silence any transposons that share base pair sequence with the piRNA

How does silencing by piRNA and Piwi proteins occur?

though DNA methylation and heterochromatin formation around the site of the transposon integration

True or False: In animals, the cells of the germ line undergo a global demethylation of their genomic DNA.

true

What can demethylation activate?

transposons that were previously silenced

What could happen if activated transposons undergo transposition?

insertional mutagensis

True or False: By silencing transposons in the germ line, piRNAs help to prevent transposons from causing heritable mutations.

true

True or False: In D. melanogaster flies of the P strain carry P element transposons in their genome.

true

How can D. melanogaster prevent transposition of P elements?

by synthesizing an inhibitor molecule

True or False: Genome comparisons of the P strain and the M strain revealed that the P strain carries - in addition to the P element transposon - genes encoding piRNAs that are complementary to mRNA transcripts from the P element transposase gene.

true

What do M strain flies lack?

both the P element and the corresponding piRNAs

What did the findings about piRNA and D. melanogaster suggest?

• The inhibitor molecule that keeps the P element from undergoing transposition in flies of the P strain is in actuality a set of piRNAs that silence the P element locus

• The P strain evolved these piRNAs genes after - and in response to - the initial insertion of the P element transposon into its genomic DNA