Shintaro Iwasaki

Hsc70/Hsp90 Chaperone Machinery Mediates ATP-Dependent RISC Loading of Small RNA Duplexes

Small silencing RNAs--small interfering RNAs (siRNAs) or microRNAs (miRNAs)--direct posttranscriptional gene silencing of their mRNA targets as guides for the RNA-induced silencing complex (RISC). Both siRNAs and miRNAs are born double stranded. Surprisingly, loading these small RNA duplexes into Argonaute proteins, the core components of RISC, requires ATP, whereas separating the two small RNA strands within Argonaute does not. Here we show that the Hsc70/Hsp90 chaperone machinery is required to load small RNA duplexes into Argonaute proteins, but not for subsequent strand separation or target cleavage. We envision that the chaperone machinery uses ATP and mediates a conformational opening of Ago proteins so that they can receive bulky small RNA duplexes. Our data suggest that the chaperone machinery may serve as the driving force for the RISC assembly pathway.

ATP-dependent human RISC assembly pathways

The assembly of RNA-induced silencing complex (RISC) is a key process in small RNA–mediated gene silencing. In humans, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are incorporated into RISCs containing the Argonaute (AGO) subfamily proteins Ago1–4. Previous studies have proposed that, unlike Drosophila melanogaster RISC assembly pathways, human RISC assembly is coupled with dicing and is independent of ATP. Here we show by careful reexamination that, in humans, RISC assembly and dicing are uncoupled, and ATP greatly facilitates RISC loading of small-RNA duplexes. Moreover, all four human AGO proteins show remarkably similar structural preferences for small-RNA duplexes: central mismatches promote RISC loading, and seed or 3′-mid (guide position 12–15) mismatches facilitate unwinding. All these features of human AGO proteins are highly reminiscent of fly Ago1 but not fly Ago2.

Drosophila Argonaute1 and Argonaute2 Employ Distinct Mechanisms for Translational Repression

microRNAs induce translational repression by binding to partially complementary sites on their target mRNAs. We have established an in vitro system that recapitulates translational repression mediated by the two Drosophila Argonaute (Ago) subfamily proteins, Ago1 and Ago2. We find that Ago1-RISC (RNA-induced silencing complex) represses translation primarily by ATP-dependent shortening of the poly(A) tail of its mRNA targets. Ago1-RISC can also secondarily block a step after cap recognition. In contrast, Ago2-RISC competitively blocks the interaction of eIF4E with eIF4G and inhibits the cap function. Our finding that the two Ago proteins in flies regulate translation by different mechanisms may reconcile previous, contradictory explanations for how miRNAs repress protein synthesis.

The microRNA pathway and cancer

MicroRNAs (miRNAs) are ∼22 nt long, non‐coding RNAs that guide post‐transcriptional gene silencing of their target genes and regulate diverse biological processes including cancer. miRNAs do not act alone, but require assembly into RNA‐induced silencing complex (RISC). In this review, we summarize how miRNAs are produced, assembled into RISC, and regulate target mRNAs, and discuss how the miRNA pathway is involved in cancer. (Cancer Sci 2010; 101: 2309–2315)

Characterization of Arabidopsis decapping proteins AtDCP1 and AtDCP2, which are essential for post‐embryonic development

Although decapping is an important process in eukaryotic mRNA turnover, little is known about this process in plants. Here, we identified Arabidopsis thaliana decapping proteins AtDCP1 and AtDCP2 and showed that (I) AtDCP2 is an active decapping enzyme, (II) AtDCP1 interacts with itself, (III) AtDCP1 and AtDCP2 are localized to cytoplasmic foci (putative Arabidopsis processing body), and (IV) AtDCP1 and AtDCP2 are essential for post‐embryonic development. Our findings provide new insights into the role of decapping‐dependent mRNA turnover.

Defining fundamental steps in the assembly of the Drosophila RNAi enzyme complex

Small RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) silence the expression of their complementary target messenger RNAs via the formation of effector RNA-induced silencing complexes (RISCs), which contain Argonaute (Ago) family proteins at their core. Although loading of siRNA duplexes into Drosophila Ago2 requires the Dicer-2–R2D2 heterodimer and the Hsc70/Hsp90 (Hsp90 also known as Hsp83) chaperone machinery, the details of RISC assembly remain unclear. Here we reconstitute RISC assembly using only Ago2, Dicer-2, R2D2, Hsc70, Hsp90, Hop, Droj2 (an Hsp40 homologue) and p23. By following the assembly of single RISC molecules, we find that, in the absence of the chaperone machinery, an siRNA bound to Dicer-2–R2D2 associates with Ago2 only transiently. The chaperone machinery extends the dwell time of the Dicer-2–R2D2–siRNA complex on Ago2, in a manner dependent on recognition of the 5′-phosphate on the siRNA guide strand. We propose that the chaperone machinery supports a productive state of Ago2, allowing it to load siRNA duplexes from Dicer-2–R2D2 and thereby assemble RISC.

Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumour cells and repress translation of specific messenger RNAs. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its messenger RNA selectivity is proposed to reflect highly structured 5′ untranslated regions that depend strongly on eIF4A-mediated unwinding. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA. Here we show that secondary structure in 5′ untranslated regions is only a minor determinant for RocA selectivity and that RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA–eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA–protein interactions.

Argonaute-mediated translational repression (and activation)

microRNAs (miRNAs) down-regulate the expression of their target genes by inducing translational repression and/or mRNA decay. Under specific conditions, miRNAs can even activate translation of their target mRNAs. These processes occur via miRNA-protein complexes, or RNA-induced silencing complexes (RISCs), which contain Argonaute (Ago) subfamily protein as a core component. However, detailed mechanisms of miRNA-mediated translational regulation remain unclear. We recently reported that, in Drosophila, both of the two Ago proteins, Ago1 and Ago2, can repress translation of the target mRNAs, but by remarkably different mechanisms. Furthermore, we here show that Ago2, but not Ago1, can activate translation of the target mRNAs when they lack the poly(A) tail, suggesting that the length of poly(A) tail is an important determinant for the consequences of Ago2 function. This review focuses on how miRNAs regulate translation in light of these new findings.

The Growing Toolbox for Protein Synthesis Studies

Protein synthesis stands at the last stage of the central dogma of molecular biology, providing a final regulatory layer for gene expression. Reacting to environmental cues and internal signals, the translation machinery can quickly tune the translatome from a pre-existing pool of RNAs, before the transcriptome changes. Although the translation reaction itself has been known since the 1950s, the quantitative or even qualitative measurement of its efficacy in cells has posed experimental and analytic hurdles. In this review, we outline the array of state-of-the-art methods that have emerged to tackle the hidden aspects of translational control.

Argobots: A Lightweight Low-Level Threading and Tasking Framework

In the past few decades, a number of user-level threading and tasking models have been proposed in the literature to address the shortcomings of OS-level threads, primarily with respect to cost and flexibility. Current state-of-the-art user-level threading and tasking models, however, either are too specific to applications or architectures or are not as powerful or flexible. In this paper, we present Argobots, a lightweight, low-level threading and tasking framework that is designed as a portable and performant substrate for high-level programming models or runtime systems. Argobots offers a carefully designed execution model that balances generality of functionality with providing a rich set of controls to allow specialization by end users or high-level programming models. We describe the design, implementation, and performance characterization of Argobots and present integrations with three high-level models: OpenMP, MPI, and colocated I/O services. Evaluations show that (1) Argobots, while providing richer capabilities, is competitive with existing simpler generic threading runtimes; (2) our OpenMP runtime offers more efficient interoperability capabilities than production OpenMP runtimes do; (3) when MPI interoperates with Argobots instead of Pthreads, it enjoys reduced synchronization costs and better latency-hiding capabilities; and (4) I/O services with Argobots reduce interference with colocated applications while achieving performance competitive with that of a Pthreads approach.