What is RNAi and why do I care?

Precise gene expression is crucial for cellular functioning and is therefore regulated at multiple levels including post-transcriptionally (after being copied from DNA to RNA) through the RNA interference (RNAi) pathway. In RNAi, small RNAs (see below) guide silencing machinery to messenger RNA (mRNA) with complementary sequences, preventing that mRNA from being turned into protein. In nature, RNAi is a fundamental biological process that plays a key role in keeping the tight regulation required to allow cells to grow and develop in a controlled manner (i.e. without becoming cancerous). Additionally, because RNAi is sequence-guided, synthetic small RNAs can be designed to target specific genes; these RNAs can thus be used to investigate the roles of various gene products in diverse biological processes and hold promise for the treatment of diseases for which the genetic cause is known.

MORE DETAILS

Small RNA (sRNA): The majority of genes transcribed in animal and plant genomes are never translated into protein – instead, these gene products function as non-coding RNAs (ncRNAs). One of the main ncRNA subtypes is small RNA (sRNA), which represses genes by directing silencing machinery to target mRNAs containing complementary sequences. sRNA includes microRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs). sRNA is typically classified in terms of where it comes from (source), how it matures (biogenesis pathway) and how it functions (effector pathway).

Source and Function

miRNAs and piRNAs are both products of the endogenous genome (the host cell’s own DNA), but they use different biogenesis and effector pathways. piRNAs are 24-26 nucleotide (nt) long, mature through a piRNA-specific biogenesis pathway, and are involved in preventing repetitive DNA sequences called transposons from “hopping around” and wreaking havoc in the genomes of cells in the germ line (cells that have the potential to be passed on to offspring). 

siRNAs and miRNAs are similar in size (~20nt) and they use the same biogenesis pathway (although siRNA enters at a later step) and effector, but they come from different sources and have different functions. miRNAs are transcribed from genes in the cell’s own genome and fold back upon themselves to form long RNA hairpins (see “sRNA biogenesis” below for more details.  In contrast, siRNAs usually come from exogenous double-stranded RNA (dsRNA) sources, such as viral transcripts or synthetic constructs (e.g. introduced by scientists for targeted gene silencing). 

miRNAs and siRNAs differ in how they interact with and silence their targets. siRNAs match their targets completely and play a prominent role in plant and invertebrate immune defense.  Unlike siRNAs, miRNAs have only partial guide/target pairing – the main determinant of miRNA target recognition is full matching of a 6nt “seed sequence” (typically within the target mRNA’s 3’ UTR), and the extent of complementarity along the rest of the miRNA differs between targets. This difference in complementarity has implications for the effector pathway (see below).

miRNAs serve as the predominant sRNAs in vertebrates and regulate mRNA. miRNA-mediated regulation allows for high specificity as well as concerted action. A single miRNA can target numerous mRNA molecules, allowing for the regulation of entire pathways and processes. At the same time, targets can have additional binding sites for more specific/rare miRNA, allowing for regulation of individual components. It is estimated that more than 60% of all protein-coding genes are regulated by miRNAs: in extreme cases, miRNA can be used to quickly produce widespread responses, but in most cases, it is believed to play more of a “fine-tuning” role, allowing for tight regulation of gene products – while this may sound minor, it can mean the difference between normal cell growth and cancer.

Effector Pathway

All 3 types of sRNA must bind to an effector molecule to carry out silencing, and this role is played by the Argonaute superfamily of proteins, which can be further divided into 3 main clades: Piwis, which interact exclusively with piRNAs; worm-specific Argonautes (wAgos); and Argonaute (Ago) Agos, which bind both miRNAs and siRNAs to form the core RNA induced silencing complex (RISC) which targets mRNAs’ “Achilles” heels to silence them. 

How does it work? After protein-coding genes are transcribed into messenger RNA (mRNA), this RNA has its ends protected by the addition of a 5’ methyl-G cap and a 3’ poly-adenosine (poly-A) tail. These modifications allow the mRNA to escape degradation machinery designed to recognize and destroy foreign RNA (e.g. viral RNA transcripts), which have “naked” ends and are therefore able to be degraded by enzymes called exonucleases. RISCs can expose naked ends of their targets by removing these end protections or cleaving the target mRNA internally to generate new ends. These ends flag the mRNA for degradation and thus allows for tight regulation of gene expression.

Which way to go? The mechanisms RISC uses depends on the Argonaute protein (e.g. whether it has “slicing” ability) and the amount of sequence complementarity between the guide miRNA and the target mRNA. When the sRNA guide matches a target exactly, as is the case with siRNAs, the Ago protein cleaves the target across from nucleotides 10 and 11 of the guide, triggering the target’s degradation by exonucleases. When the sRNA doesn’t match the target completely, as with miRNA, Argonaute can’t cleave the target, so other co-factors are required to trigger silencing. Specifically, a scaffolding protein called GW182 binds to Ago and recruits silencing factors: deadenylation complexes remove the 3’ tail and a decapping complex removes the 5’ cap, exposing the ends to exonucleases.

In addition to promoting mRNA decay, RISC has been reported to repress targets by: preventing translation initiation and elongation; triggering premature termination and co-translational protein degradation; sequestering targets from translational machinery; and inhibiting transcription by directing chromatin remodeling. These diverse mechanisms all depend on the ability of miRNA-loaded Ago to recognize and bind its targets, which is why I’m so interested in Ago!.

TYPEmiRNAsiRNApiRNA
SOURCEendogenous short
RNA hairpins
long dsRNA
(typically exogenous)
endogenous ssRNA
EFFECTORAgoAgoPIWI
FUNCTIONtranslational regulation
in vertebrates
viral defense in
invertebrates
transposon repression
in germ line

EVEN MORE DETAILS:

sRNA Biogenesis: miRNA production requires several concerted steps: primary miRNAs (pri-miRNAs) are transcribed in the nucleus and fold back onto themselves as hairpins. Some are transcribed individually, while others are transcribed from the same promoter as polycistronic transcripts and some (mirtrons) are transcribed from the introns of other genes. In the conventional pathway, pri-miRNA, regardless of the source, is trimmed in the nucleus by a microprocessor complex composed of the RNAseIII-family endonuclease Drosha and its cofactor DGCR8 to form a ~60-70nt hairpin with a 2nt 3’ overhang. This pre-miRNA is transported through the nuclear pore complex (NPC) via exportin 5 (Xpo5) into the cytoplasm, where another RNAseIII-family enzyme, Dicer, with its cofactor TRBP or PACT, further trims it to a ~20nt miRNA duplex (pre-siRNAs also enter the pathway at this step). This duplex is then loaded into Ago with the help of Hsp90/Hsc70 chaperone machinery to form the “pre-RISC” and the passenger strand is released to form active RISC. This conventional maturation pathway is required for the vast majority of miRNAs, but there are exceptions.

VISUAL SUMMARY: