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It remains unclear how post-transcriptional gene silencing in plants discriminates aberrant RNAs from canonical mRNAs. Now, a study shows that the poly(A) tail of canonical mRNAs blocks RDR6 from converting them into the substrates for gene silencing.

The assembly of single Drosophila RNA-induced silencing complexes (RISCs) is reconstituted using seven purified proteins, revealing that chaperones help stabilize the interaction of the protein heterodimer Dicer-2–R2D2 bound to the short interfering RNA with Ago2.

Fly Dicer-2 is thought to use two distinct – processive or distributive – modes of cleavage by distinguishing the terminal structures of double-stranded RNA (dsRNA) substrates with the help of its cofactor LoquaciousPD (Loqs-PD). Here the authors show by single-molecule imaging that dsRNA terminal structures and Loqs-PD change the probability for Dicer to initiate processive cleavage but not the mode of cleavage action per se.

RNA silencing is a sequence-specific gene regulation system conserved in eukaryotes, at the core of which lies the Argonaute protein family. Crystallographic studies of eukaryotic Argonaute proteins now reveal remarkably similar overall structures to their prokaryotic homologs while shedding new light on the fundamental relationship between their conformational dynamics and sophisticated strategies to silence specific targets.

A silkworm model recapitulates key steps of Zucchini-mediated cleavage of pre-pre-piRNA and provides insights into Zucchini-mediated and -independent pathways that generate pre-piRNAs, which converge to a common piRNA maturation step.

miRNAs are loaded onto Argonautes (Agos) to guide silencing of targets, but duplex unwinding is required for targeting. Detection of Drosophila Ago1 complexes containing the duplexed or unwound miRNA now give insight into the basis for cleavage-independent unwinding of miRNA duplexes to generate a functional, mature complex.

Small RNAs function within the context of RNA-induced silencing complexes (RISCs) containing Argonaute (AGO) subfamily proteins. Experiments now show that human RISC assembly is uncoupled from dicing and is facilitated by ATP to load the small RNA duplexes but is not necessary to unwind them. The four human AGO proteins show no obvious structural preferences for small RNA duplexes in contrast to the situation in flies and worms where small RNAs are actively sorted into distinct AGO proteins according to their structural features.

Small noncoding RNAs function together with Argonaute (Ago) proteins as part of RNA-induced silencing complexes (RISCs). New biochemical analyses have identified the N domain of human AGO2 as the initiator of small duplex RNA unwinding during RISC assembly, which is required for both slicer-dependent and slicer-independent unwinding mechanisms.

It has been unclear how fly Dicer-1 exclusively recognizes pre-miRNAs. New analyses show that fly Dicer-1 recognizes the single-stranded terminal loop structure of pre-miRNAs through its N-terminal helicase domain, checks the loop size and measures the distance between the 3′ overhang and the terminal loop. This unique mechanism allows fly Dicer-1 to inspect the authenticity of pre-miRNA structures.

Cryo-electron microscopy structures of Drosophila Dicer-2–R2D2 complexes with and without small interfering RNA reveal how the RNA is presented to Argonaute in the correct orientation for viral gene silencing.

A global analysis of translation efficiency and RNA accumulation revealed that microRNAs in the unicellular green alga Chlamydomonas regulate target gene expression via RNA destabilization and translational repression like land plants and metazoans, yet in a different way.

The evolutionarily conserved RNA-binding protein GTSF1 and its homologues interact with members of the PIWI class of Argonaute proteins, increasing the efficiency of the RNA-cleaving activity of PIWI proteins, an essential function across the animal kingdom.

Mature piRNAs are produced from long single-stranded precursor RNAs. Here the authors show that RNase κ generates 2′,3′-cyclic phosphate-containing piRNA precursors and promotes piRNA production.