Structural Biochemistry/Nucleic Acid/RNA/Structural insights into RNA interference (RNAi)

Introduction

RNA interference (RNAi) are small RNA molecules that regulate eukaryotic gene expression. This first starts with the production of ~20-30 nucleotide RNAs sequences, microRNAs (miRNAs) or small interfering RNAs (siRNAs), that have the potential of forming base pair with parts of the mRNA. The miRNAs and siRNAs then assemble into large mutliprotein effectors, molecules that bind to and alter the activity of that protein, called RNA-induced silencing complexes (RISCs) that bind to and initiate target transcript’s destruction.

miRNAs and siRNAs are initially produced as long stem-loop structures in the nucleus through endonucleolytic processing of double-stranded DNA. miRNA is recognized by DGCR8, a protein which exports the RNA to the RNase III-family endonuclease Drosha for initial double-stranded cleavage. After cleavage, the pre-miRNA is cleaved by a second RNase III-family endonuclease called Dicer, in the cytoplasm by Exportin 5. siRNA is finally produced by a Dicer-mediated cleavage of long double-stranded RNAs, resulting in a duplex that contains an antisense guide strand, and the sense or passenger strand.

miRNAs and siRNAs are efficiently incorporation into other RISC complex due to their specific length of the RNA duplex, and the characteristic phosphate carrying 5’ group and their dinucleotide overhang 3’ group. Structural analysis of prokaryotic RNase III and a eukaryotic Dicer enzyme have shown how these critical features of siRNAs and miRNAs are generated. Agronaute (Ago), a core component of RISCs, is what allows miRNAs and siRNAs to attach to RISCs and help trigger the destruction of certain transcript. A guiding strand brings miRNAs and siRNAs Ago, followed by the descruction/dissociation of the miRNAs’ and siRNAs’ passenger strand. After their attachment, the RISCs use the small RNA’s sequence as guides to figure out which specific sequence to either repress the translation of, or to induce their degradation. Specifically, the RISCs target the RNA sequences that are complementary to the small RNAs’ sequence. This process is further modified by RITS, a RNA-induced transcriptional silencing complex which mediates the transcriptionalgene silencing by promoting heterochromatin formations. Piwi-interacting RNAs, piRNAs, are also involved in this process.

Piwi-interacting RNA (piRNA)

Piwi-interacting RNAs, or piRNAs, are a class of small RNAs that are expressed in eukaryotic cells. piRNA function to support germ line development, although recent studies have suggested it may be extended to that of somatic ovarian tissues. It also contributes to the direct silencing of genes.

Along with miRNA and siRNA, piRNA is a constituent of non-coding RNA molecules. However, their biogenesis pathway is distinct from that of miRNA and siRNA.

piRNA undergoes an amplification pathway that is referred to as the 'Ping Pong' mechanism. Primary piRNAs, most likely generated from transcription of long single-stranded RNAs, cause the recruitment of piwi proteins upon recognizing their complementary targets. Then the transcript is cleaved 10 nucleotides from the 5' end to produce a secondary piRNA that targets adenine at the 10th position.

RISC loading

In order to be efficient, mature miRNA and siRNA duplexes have to be transferred from the Dicer cleaving protein to Argonaute in order to form an active RISC. The components required for this process is a Dicer complexed with a dsRBP cofactor, TRBP, and a mature siRNA duplex.

While the exact process of how siRNA is transferred from the Dicer to the Argonaute is not yet known, what is known for certain is that this process involves the selection of the guide strand and the removal of the passenger strand. However, recently, analysis via negative-strain electron microscopy of the human RISC loading complex (RLC) and its components have provided a structural framework for ideas on testing exactly how siRNA duplexes can be passed from Dicer-TRBP to Ago2 during RISC loading. These analysis suggest that TRBP interacts flexibly with Dicer’s helicase domain and that Ago2 forms a closed complex of particles by stretching between TRBP and Dicer. Using previous biochemical studies, this Ago2-Dicer interaction is believed to occur between the catalytic PIWI domain of Ago2 and the RNase IIIa domain of Dicer.

Mechanisms of RNA silencing

After the RISC complex is loaded, the Ago-guide strand complex can now bind to its target mRNA. This occurs through the base pairing of the guide strand with sequences in the 3’-untranslated region, called UTR, of the mRNA. Only when there is as full guide-target strand complementarity does the common mechanism for RNA silencing by siRNAs via endonucleolytic ‘slicer’ activity of Ago start the target strand cleavage. There are two distinctive domains in the Ago family of proteins that can bind to the guide strand of the RISC complex: the PAZ domain and the RNase H-like PIWI domain. the PAZ domain binds to the 3’-end of the guide strand and the RNase H-like PIWI domain houses the slicing active site. A small portion of the Ago proteins also contain a MID domain. the MID domain forms the binding pocket for the 5’-phosphate of the guide strand by hydrogen bonding extensively with conserved residues and a Mg2+ ion.