Yuichiro Mishima

A Novel miRNA Processing Pathway Independent of Dicer Requires Argonaute2 Catalytic Activity

No Dicer for Me MicroRNAs (miRNAs) are small noncoding RNAs found in most eukaryotes. Most are processed from primary transcripts in the nucleus by the microprocessor enzyme complex, which includes the nuclease Drosha, with a small number being generated by the messenger RNA splicing machinery. All pre-miRNAs are then exported into the cytoplasm where they are cleaved further by a second nuclease, Dicer, into the mature, functional miRNA. Cifuentes et al. (p. 1694, published online 6 May), now show that in a Dicer mutant fish at least one miRNA, miR-451, is still formed from pre-miR-451. The processing of pre-miR-451 requires the slicing activity of another protein in the miRNA pathway, Argonaute2. The unusual secondary structure of the pre-miR-451 determines its noncanonical processing pathway, which suggests that other miRNAs might also be processed in this way. The unusual secondary structure of a precursor microRNA determines its noncanonical processing. Dicer is a central enzyme in microRNA (miRNA) processing. We identified a Dicer-independent miRNA biogenesis pathway that uses Argonaute2 (Ago2) slicer catalytic activity. In contrast to other miRNAs, miR-451 levels were refractory to dicer loss of function but were reduced in MZago2 (maternal-zygotic) mutants. We found that pre-miR-451 processing requires Ago2 catalytic activity in vivo. MZago2 mutants showed delayed erythropoiesis that could be rescued by wild-type Ago2 or miR-451-duplex but not by catalytically dead Ago2. Changing the secondary structure of Dicer-dependent miRNAs to mimic that of pre-miR-451 restored miRNA function and rescued developmental defects in MZdicer mutants, indicating that the pre-miRNA secondary structure determines the processing pathway in vivo. We propose that Ago2-mediated cleavage of pre-miRNAs, followed by uridylation and trimming, generates functional miRNAs independently of Dicer.

Zebrafish miR-1 and miR-133 shape muscle gene expression and regulate sarcomeric actin organization

microRNAs (miRNAs) represent approximately 4% of the genes in vertebrates, where they regulate deadenylation, translation, and decay of the target messenger RNAs (mRNAs). The integrated role of miRNAs to regulate gene expression and cell function remains largely unknown. Therefore, to identify the targets coordinately regulated by muscle miRNAs in vivo, we performed gene expression arrays on muscle cells sorted from wild type, dicer mutants, and single miRNA knockdown embryos. Our analysis reveals that two particular miRNAs, miR-1 and miR-133, influence gene expression patterns in the zebrafish embryo where they account for >54% of the miRNA-mediated regulation in the muscle. We also found that muscle miRNA targets (1) tend to be expressed at low levels in wild-type muscle but are more highly expressed in dicer mutant muscle, and (2) are enriched for actin-related and actin-binding proteins. Loss of dicer function or down-regulation of miR-1 and miR-133 alters muscle gene expression and disrupts actin organization during sarcomere assembly. These results suggest that miR-1 and miR-133 actively shape gene expression patterns in muscle tissue, where they regulate sarcomeric actin organization.

DAZL Relieves miRNA-Mediated Repression of Germline mRNAs by Controlling Poly(A) Tail Length in Zebrafish

Background During zebrafish embryogenesis, microRNA (miRNA) miR-430 contributes to restrict Nanos1 and TDRD7 to primordial germ cells (PGCs) by inducing mRNA deadenylation, mRNA degradation, and translational repression of nanos1 and tdrd7 mRNAs in somatic cells. The nanos1 and tdrd7 3′UTRs include cis-acting elements that allow activity in PGCs even in the presence of miRNA-mediated repression. Methodology/Principal Findings Using a GFP reporter mRNA that was fused with tdrd7 3′UTR, we show that a germline-specific RNA-binding protein DAZ-like (DAZL) can relieve the miR-430-mediated repression of tdrd7 mRNA by inducing poly(A) tail elongation (polyadenylation) in zebrafish. We also show that DAZL enhances protein synthesis via the 3′UTR of dazl mRNA, another germline mRNA targeted by miR-430. Conclusions/Significance Our present study indicated that DAZL acts as an “anti-miRNA factor” during vertebrate germ cell development. Our data also suggested that miRNA-mediated regulation can be modulated on specific target mRNAs through the poly(A) tail control.

MicroRNAs Trigger Dissociation of eIF4AI and eIF4AII from Target mRNAs in Humans

In animals, key functions of microRNA-induced silencing complex (miRISC) are translational repression and deadenylation followed by mRNA decay. While miRISC represses translation initiation, it is poorly understood how miRISC exerts this function. Here we assessed the effect of miRISC on synergistic recruitment of translation initiation factors to target mRNAs by using direct biochemical assays. We show that miRISC promotes eIF4AI and eIF4AII release from target mRNAs prior to dissociation of eIF4E and eIF4G in a deadenylation-independent manner. Strikingly, miRISC-induced release of eIF4AI and eIF4AII from target mRNAs and miRISC-induced inhibition of cap-dependent translation can both be counteracted by the RNA-binding protein HuD via a direct interaction of HuD with eIF4A. Furthermore, the pharmacological eIF4A inhibitor silvestrol, which locks eIF4A on mRNAs, conferred resistance to miRNA-mediated translational repression. In summary, we propose that both eIF4AI and eIF4AII are functionally important targets in miRISC-mediated translation control.

miR-1 and miR-206 regulate angiogenesis by modulating VegfA expression in zebrafish

Cellular communication across tissues is an essential process during embryonic development. Secreted factors with potent morphogenetic activity are key elements of this cross-talk, and precise regulation of their expression is required to elicit appropriate physiological responses. MicroRNAs (miRNAs) are versatile post-transcriptional modulators of gene expression. However, the large number of putative targets for each miRNA hinders the identification of physiologically relevant miRNA-target interactions. Here we show that miR-1 and miR-206 negatively regulate angiogenesis during zebrafish development. Using target protectors, our results indicate that miR-1/206 directly regulate the levels of Vascular endothelial growth factor A (VegfA) in muscle, controlling the strength of angiogenic signaling to the endothelium. Conversely, reducing the levels of VegfAa, but not VegfAb, rescued the increase in angiogenesis observed when miR-1/206 were knocked down. These findings uncover a novel function for miR-1/206 in the control of developmental angiogenesis through the regulation of VegfA, and identify a key role for miRNAs as regulators of cross-tissue signaling.

Translational inhibition by deadenylation-independent mechanisms is central to microRNA-mediated silencing in zebrafish

MicroRNA (miRNA) is a class of small noncoding RNA approximately 22 nt in length. Animal miRNA silences complementary mRNAs via translational inhibition, deadenylation, and mRNA degradation. However, the underlying molecular mechanisms remain unclear. A key question is whether these three outputs are independently induced by miRNA through distinct mechanisms or sequentially induced within a single molecular pathway. Here, we successfully dissected these intricate outputs of miRNA-mediated repression using zebrafish embryos as a model system. Our results indicate that translational inhibition and deadenylation are independent outputs mediated by distinct domains of TNRC6A, which is an effector protein in the miRNA pathway. Translational inhibition by TNRC6A is divided into two mechanisms: PAM2 motif-mediated interference of poly(A)-binding protein (PABP), and inhibition of 5′ cap- and poly(A) tail-independent step(s) by a previously undescribed P-GL motif. Consistent with these observations, we show that, in zebrafish embryos, miRNA inhibits translation of the target mRNA in a deadenylation- and PABP-independent manner at early time points. These results indicate that miRNA exerts multiple posttranscriptional outputs via physically and functionally independent mechanisms and that direct translational inhibition is central to miRNA-mediated repression.

Elements and machinery of non-coding RNAs: toward their taxonomy

Although recent transcriptome analyses have uncovered numerous non‐coding RNAs (ncRNAs), their functions remain largely unknown. ncRNAs assemble with proteins and operate as ribonucleoprotein (RNP) machineries, formation of which is thought to be determined by specific fundamental elements embedded in the primary RNA transcripts. Knowledge about the relationships between RNA elements, RNP machinery, and molecular and physiological functions is critical for understanding the diverse roles of ncRNAs and may eventually allow their systematic classification or “taxonomy.” In this review, we catalog and discuss representative small and long non‐coding RNA classes, focusing on their currently known (and unknown) RNA elements and RNP machineries.

miR-1-2 Gets to the Heart of the Matter

Although many microRNAs (miRNAs) and their targets have been identified, the importance of miRNAs in vivo is still unclear. In this issue, Zhao et al. (2007) generate mice deficient in a cardiac-specific miRNA, miR-1-2, and reveal that this microRNA plays a crucial role in heart development and physiology.

Roles of mRNA Fate Modulators Dhh1 and Pat1 in TNRC6-dependent Gene Silencing Recapitulated in Yeast

Background: Animal microRNAs silence their target mRNAs by promoting mRNA degradation and inhibiting translation via GW182/TNRC6. Results: TNRC6 induces silencing effects in S. cerevisiae via CCR4-NOT complex and Dhh1-Pat1 when tethered to reporter mRNAs. Conclusion: TNRC6 utilizes the conserved mRNA fate modulators for gene silencing in yeast. Significance: Yeast genetic tools are now available to study intricate actions of TNRC6. The CCR4-NOT complex, the major deadenylase in eukaryotes, plays crucial roles in gene expression at the levels of transcription, mRNA decay, and protein degradation. GW182/TNRC6 proteins, which are core components of the microRNA-induced silencing complex in animals, stimulate deadenylation and repress translation via recruitment of the CCR4-NOT complex. Here we report a heterologous experimental system that recapitulates the recruitment of CCR4-NOT complex by TNRC6 in S. cerevisiae. Using this system, we characterize conserved functions of the CCR4-NOT complex. The complex stimulates degradation of mRNA from the 5′ end by Xrn1, in a manner independent of both translation and deadenylation. This degradation pathway is probably conserved in miRNA-mediated gene silencing in zebrafish. Furthermore, the mRNA fate modulators Dhh1 and Pat1 redundantly stimulate mRNA decay, but both factors are required for poly(A) tail-independent translation repression by tethered TNRC6A. Our tethering-based reconstitution system reveals that the conserved architecture of Not1/CNOT1 provides a binding surface for TNRC6, thereby connecting microRNA-induced silencing complex to the decapping machinery as well as the translation apparatus.

Pervasive yet nonuniform contributions of Dcp2 and Cnot7 to maternal mRNA clearance in zebrafish

mRNA degradation is a fundamental biological process that erases transcribed genetic information from cells. During maternal‐to‐zygotic transition of animal development, thousands of maternal mRNAs are degraded by multiple mechanisms including microRNAs and codon‐mediated decay. Enzymatic requirements for maternal mRNA clearance, however, are not fully understood. Here, we analyzed a contribution of the decapping enzyme Dcp2 to maternal mRNA clearance in zebrafish by over‐expressing catalytically inactive Dcp2 and performing RNA‐seq analysis. As expected, Dcp2 had a widespread role in maternal mRNA clearance. Interestingly, each mRNA showed differential dependency on Dcp2‐mediated decapping and Cnot7‐mediated deadenylation for degradation. Correlation analysis identified several mRNA features that were associated with the observed differential dependency. Our results show pervasive yet nonuniform contributions of the decapping enzyme Dcp2 and the deadenylase Cnot7 to maternal mRNA clearance.