RNA Interference (RNAi) Pathway Craig Mello Andrew Fire - In 2006 the Nobel Prize in Medicine was awarded to Craig Mello and Andrew Fire for their discovery and elucidation of the RNA interference or RNAi pathway. In a nutshell RNAi is the process by which mRNA transcripts are either degraded or prevented from being translated. At its most basic form a segment of double stranded RNA is cleaved into relatively small (21-24nt) fragments by an enzyme called Dicer. The resulting pieces are called short interfering RNAs or siRNAs for short. These short double stranded siRNAs are then bound by a complex of proteins that are called RISC (RNA induced silencing complex). This complex unwinds the two strands. One strand is discarded while the other is retained. The RISC –RNA complex then searches for complementary sequences within messenger RNAs and forms localized regions of double stranded RNA. The formation of siRNA-mRNA complexes triggers the destruction of the mRNA transcript which of course then prevents translation. In some circumstances the mRNA transcript is not degraded but simply prevented from being translated. This pathway appears to be present in all metazoans and is used to regulated gene expression (after transcriptional has taken place).

The microRNA (miRNA) Pathway and the Regulation of Gene Expression - microRNAs (miRNAs) are one type of siRNAs that are involved in the silencing of gene expression. In most cases, gene expression is down-regulated and not eliminated therefore microRNAs are thought to function in the “fine-tuning” of gene expression. - Most metazoan genomes are predicted to encode hundreds to thousands of microRNA genes (the exact number depends upon the organism/genome in question). These miRNA genes are transcribed by RNA Pol II (remember that this is the same enzyme that transcribe protein coding genes. - The initial transcript (pri-miRNA) are usually ~500 bases in size and folded into a single stem loop with trailing tails. The pri-miRNA is then cleaved into a smaller ~70nt stem loop pre-miRNA fragment by two nuclear enzymes called Pasha and Drosha. - The pre-miRNA is exported into the cytoplasm by a nuclear export factor called Exportin-5. - Once the pre-miRNA is transported to the cytoplasm the Dicer enzyme then cleaves the loop section leaving a 21-24nt double-stranded RNA fragment (miRNA). This fragment is then bound by members of the RISC complex. One important component on this complex is an RNA binding protein called Argonaute (Ago). - Within the RISC complex the two miRNA strands are separated (one is degraded and one is retained). The RISC-miRNA complex then searches for complementary sequences within the messenger RNA transcripts that are present within the cell. - If the region of complementarity between the miRNA and the mRNA transcript is perfect then the transcript will be degraded by the RISC complex. If there is only partial homology between the two fragments then the RISC complex will prevent translation without degrading the transcript.

miRNA:mRNA duplexes Perfect vs Imperfect Matches - The most common position for microRNAs to bind within the mRNA transcript is the 3`UTR. However, miRNAs have been shown to bind to other positions as well. The binding of the miRNA to complementary sequences within the 3`UTR can lead to either degradation of the mRNA or to just a block in translation. The most important determinant is whether or not the first 7-8 bases (called the seed region) within the miRNA have perfect or imperfect homology with the mRNA transcript. If the homology is perfect then the transcript will be degraded (top and bottom left). If the homology is less than perfect then the transcript will survive and translation will be blocked instead (top and bottom right). The degree of homology between the remaining 13-14 bases of the miRNA and the mRNA transcript is not a factor in determining whether or not the transcript will be degraded. - A single mRNA transcript can be bound by multiple miRNAs. Many mRNA transcripts will contain multiple binding sites for a single type of miRNA. Additionally, several different types of miRNAs can simultaneously bind to a single mRNA transcript. The regulation of an single transcript is therefore dependent upon the combinatorial code of miRNAs that bind to the 3`UTR. The length of the mRNA 3`UTR is a factor in how many different miRNAs can be bound to the transcript. mRNA transcripts with long 3`UTR segments can be bound by more miRNAs than mRNA transcripts with very short 3`UTR segments. - A single type of miRNA can bind to multiple different mRNA transcripts thereby regulating the expression of multiple genes.

Mechanisms of miRNA Activity - In the previous slides we have discussed that miRNAs can either degrade mRNA transcripts or inhibit translation. Here we discuss several experimentally verified models for how miRNAs accomplish these tasks. - Earlier in the semester (while we were discussing translation) we talked about the fact that Poly A Binding Protein (PABP), which binds to the 3` end of the transcript, interacts with several eukaryotic initiation factors (eIF4E and eIF4G) which themselves are bound to the 5` end of the transcript (first panel). The formation of this circular mRNA species improves the stability of the transcript and allows higher levels of translation. - As miRNAs bind to the 3` end of the transcript the accompanying Ago protein (part of RISC) displaces the initiation factors and instead binds to the 5` end of the transcript. Without the initiation factors, the ribosome cannot initiate translation. This is called inhibiting translation initiation (second panel. - A second mechanism is that the recruitment of the miRNA-Ago complex to the mRNA transcript allows for the Ago protein to displace the ribosome from the transcript. The shortened proteins are then degraded. This mechanisms is referred to as post-initiation inhibition (third panel). - And finally, the degradation of the transcript can occur when the Ago protein recruits RNA specific nucleases. The enzymes will digest the messenger RNA transcript (fourth panel)

Using RNAi to Examine Homeotic Gene Function - In Drosophila the proboscipedia (pb) gene is one of five genes that make up the Antennapedia Hox cluster. pb is expressed in the anterior segments of the embryo that will eventually give rise to parts of the proboscis (bottom). Mutations that eliminate the activity of pb lead to the transformation of parts of the proboscis into two legs (top left panels). An analysis of the milkweed bug genome indicates that all eight Hox genes that are present in the fruit fly are also present within the milkweed bug. pb loss-of-function mutants (like the ones that exist in Drosophila) do not exist yet in the milkweed bug. - How can you determine if the pb gene plays a similar role in establishing the identity of the proboscis of the milkweed bug? You can inject short double stranded RNAs that have sequences complementary to the pb mRNA into the developing milkweed bug embryo. The RNAi pathway processes the dsRNA as described in these slides and will target the pb mRNA. The levels of pb protein will be reduced due to degradation of the transcript. Reductions of pb in the milkweed bug lead to similar proboscis-leg transformations (bottom left panels). This indicates that the role of pb has been conserved between Drosophila and the milkweed bug

Molecular Biology Study Questions - How do microRNAs inhibit gene expression? - Within which cellular compartment do miRNAs inhibit gene expression? - What will happen if there is a perfect match between the miRNA and the mRNA transcript? - What will happen if there is an imperfect match between the miRNA and the mRNA transcript? - What are the roles of the Drosha and Pashe enzymes? - What is the role of the Dicer enzyme? - What is the role of Argonaute and the RIS complex (RISC)? - What part of the mRNA transcript is most commonly targeted by miRNAs? - What would happen if a miRNA targeted the poly-A tail of mRNA transcripts? - Can a single miRNA bind to several different messenger RNA transcripts? - How does Argonaute inhibit translation? - How does Argonaute degrade messenger RNAs?

Preview of Upcoming Lecture Topics to be Covered Next Time The Hippo Tumor Suppressor Pathway Tumor Suppressors and the miRNA Pathway Weekly Article(s) “How Cells Clean House” “DIY Chromosomes” “Monogamouse”