LIN41 Post-transcriptionally Silences mRNAs by Two Distinct and Position-Dependent Mechanisms.

The RNA-binding protein (RBP) LIN41, also known as LIN-41 or TRIM71, is a key regulator of animal development, but its physiological targets and molecular mechanism of action are largely elusive. Here we find that this RBP has two distinct mRNA-silencing activities. Using genome-wide ribosome profiling, RNA immunoprecipitation, and in vitro-binding experiments, we identify four mRNAs, each encoding a transcription factor or cofactor, as direct physiological targets of C. elegans LIN41. LIN41 silences three of these targets through their 3' UTRs, but it achieves isoform-specific silencing of one target, lin-29A, through its unique 5' UTR. Whereas the 3' UTR targets mab-10, mab-3, and dmd-3 undergo transcript degradation, lin-29A experiences translational repression. Through binding site transplantation experiments, we demonstrate that it is the location of the LIN41-binding site that specifies the silencing mechanism. Such position-dependent dual activity may, when studied more systematically, emerge as a feature shared by other RBPs.

Structural basis of siRNA recognition by TRBP double-stranded RNA binding domains.

The accurate cleavage of pre-micro(mi)RNAs by Dicer and mi/siRNA guide strand selection are important steps in forming the RNA-induced silencing complex (RISC). The role of Dicer binding partner TRBP in these processes remains poorly understood. Here, we solved the solution structure of the two N-terminal dsRNA binding domains (dsRBDs) of TRBP in complex with a functionally asymmetric siRNA using NMR, EPR, and single-molecule spectroscopy. We find that siRNA recognition by the dsRBDs is not sequence-specific but rather depends on the RNA shape. The two dsRBDs can swap their binding sites, giving rise to two equally populated, pseudo-symmetrical complexes, showing that TRBP is not a primary sensor of siRNA asymmetry. Using our structure to model a Dicer-TRBP-siRNA ternary complex, we show that TRBP's dsRBDs and Dicer's RNase III domains bind a canonical 19 base pair siRNA on opposite sides, supporting a mechanism whereby TRBP influences Dicer-mediated cleavage accuracy by binding the dsRNA region of the pre-miRNA during Dicer cleavage.

Extensive oscillatory gene expression during C. elegans larval development.

Oscillations are a key to achieving dynamic behavior and thus occur in biological systems as diverse as the beating heart, defecating worms, and nascent somites. Here we report pervasive, large-amplitude, and phase-locked oscillations of gene expression in developing C. elegans larvae, caused by periodic transcription. Nearly one fifth of detectably expressed transcripts oscillate with an 8 hr period, and hundreds change >10-fold. Oscillations are important for molting but occur in all phases, implying additional functions. Ribosome profiling reveals that periodic mRNA accumulation causes rhythmic translation, potentially facilitating transient protein accumulation as well as coordinated production of stable, complex structures such as the cuticle. Finally, large-amplitude oscillations in RNA sampled from whole worms indicate robust synchronization of gene expression programs across cells and tissues, suggesting that these oscillations will be a powerful new model to study coordinated gene expression in an animal.

Bayesian prediction of RNA translation from ribosome profiling.

Ribosome profiling via high-throughput sequencing (ribo-seq) is a promising new technique for characterizing the occupancy of ribosomes on messenger RNA (mRNA) at base-pair resolution. The ribosome is responsible for translating mRNA into proteins, so information about its occupancy offers a detailed view of ribosome density and position which could be used to discover new translated open reading frames (ORFs), among other things. In this work, we propose Rp-Bp, an unsupervised Bayesian approach to predict translated ORFs from ribosome profiles. We use state-of-the-art Markov chain Monte Carlo techniques to estimate posterior distributions of the likelihood of translation of each ORF. Hence, an important feature of Rp-Bp is its ability to incorporate and propagate uncertainty in the prediction process. A second novel contribution is automatic Bayesian selection of read lengths and ribosome P-site offsets (BPPS). We empirically demonstrate that our read length selection technique modestly improves sensitivity by identifying more canonical and non-canonical ORFs. Proteomics- and quantitative translation initiation sequencing-based validation verifies the high quality of all of the predictions. Experimental comparison shows that Rp-Bp results in more peptide identifications and proteomics-validated ORF predictions compared to another recent tool for translation prediction.

The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing.

Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations co-sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT). Fractionation and membrane co-immune precipitations further confirm that siRNA-loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC-associated double-stranded siRNA, diagnostic of RISC loading, and RISC-mediated mRNA cleavage products exclusively co-sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA-independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA-mediated RNA silencing.

Transcriptome-wide measurement of ribosomal occupancy by ribosome profiling.

Gene expression profiling provides a tool to analyze the internal states of cells or organisms, and their responses to perturbations. While global measurements of mRNA levels have thus been widely used for many years, it is only through the recent development of the ribosome profiling technique that an analogous examination of global mRNA translation programs has become possible. Ribosome profiling reveals which RNAs are being translated to what extent and where the translated open reading frames are located. In addition, different modes of translation regulation can be distinguished and characterized. Here, we present an optimized, step-by-step protocol for ribosome profiling. Although established in Caenorhabditis elegans, our protocol and optimization approaches should be equally usable for other model organisms or cell culture with little adaptation. Next to providing a protocol, we compare two different methods for isolation of single ribosomes and two different library preparations, and describe strategies to optimize the RNase digest and to reduce ribosomal RNA contamination in the libraries. Moreover, we discuss bioinformatic strategies to evaluate the quality of the data and explain how the data can be analyzed for different applications. In sum, this article seeks to facilitate the understanding, execution, and optimization of ribosome profiling experiments.

Autoregulation of the nonsense-mediated mRNA decay pathway in human cells.

Nonsense-mediated mRNA decay (NMD) is traditionally portrayed as a quality-control mechanism that degrades mRNAs with truncated open reading frames (ORFs). However, it is meanwhile clear that NMD also contributes to the post-transcriptional gene regulation of numerous physiological mRNAs. To identify endogenous NMD substrate mRNAs and analyze the features that render them sensitive to NMD, we performed transcriptome profiling of human cells depleted of the NMD factors UPF1, SMG6, or SMG7. It revealed that mRNAs up-regulated by NMD abrogation had a greater median 3'-UTR length compared with that of the human mRNAome and were also enriched for 3'-UTR introns and uORFs. Intriguingly, most mRNAs coding for NMD factors were among the NMD-sensitive transcripts, implying that the NMD process is autoregulated. These mRNAs all possess long 3' UTRs, and some of them harbor uORFs. Using reporter gene assays, we demonstrated that the long 3' UTRs of UPF1, SMG5, and SMG7 mRNAs are the main NMD-inducing features of these mRNAs, suggesting that long 3' UTRs might be a frequent trigger of NMD.

A branched heterochronic pathway directs juvenile-to-adult transition through two LIN-29 isoforms.

Robust organismal development relies on temporal coordination of disparate physiological processes. In Caenorhabditis elegans, the heterochronic pathway controls a timely juvenile-to-adult (J/A) transition. This regulatory cascade of conserved proteins and small RNAs culminates in accumulation of the transcription factor LIN-29, which triggers coordinated execution of transition events. We report that two LIN-29 isoforms fulfill distinct functions. Functional specialization is a consequence of distinct isoform expression patterns, not protein sequence, and we propose that distinct LIN-29 dose sensitivities of the individual J/A transition events help to ensure their temporal ordering. We demonstrate that unique isoform expression patterns are generated by the activities of LIN-41 for lin-29a, and of HBL-1 for lin-29b, whereas the RNA-binding protein LIN-28 coordinates LIN-29 isoform activity, in part by regulating both hbl-1 and lin-41. Our findings reveal that coordinated transition from juvenile to adult involves branching of a linear pathway to achieve timely control of multiple events.

LSM2-8 and XRN-2 contribute to the silencing of H3K27me3-marked genes through targeted RNA decay.

In fission yeast and plants, RNA processing and degradation contribute to heterochromatin silencing, alongside conserved pathways of transcriptional repression. It has not been known whether similar pathways exist in metazoans. Here, we describe a pathway of silencing in Caenorhabditis elegans somatic cells, in which the highly conserved RNA-binding complex LSM2-8 contributes selectively to the repression of heterochromatic reporters and endogenous genes bearing the Polycomb mark, histone H3K27me3. This acts by degrading selected transcripts through the XRN-2 exoribonuclease. Disruption of the LSM2-8 pathway leads to mRNA stabilization. Unlike previously described pathways of heterochromatic RNA degradation, LSM2-8-mediated RNA degradation does not target nor require H3K9 methylation. Intriguingly, loss of this pathway coincides with a localized reduction in H3K27me3 at lsm-8-sensitive loci. We have thus uncovered a mechanism of RNA degradation that selectively contributes to the silencing of a subset of H3K27me3-marked genes, revealing a previously unrecognized layer of post-transcriptional control in metazoan heterochromatin.

let-7 coordinates the transition to adulthood through a single primary and four secondary targets.

The juvenile-to-adult (J/A) transition, or puberty, is a period of extensive changes of animal body morphology and function. The onset of puberty is genetically controlled, and the let-7 miRNA temporally regulates J/A transition events in nematodes and mammals. Here, we uncover the targets and downstream pathways through which Caenorhabditis elegans let-7 controls male and female sexual organ morphogenesis and skin progenitor cell fates. We find that let-7 directs all three processes by silencing a single target, the post-transcriptional regulator lin-41 In turn, the RNA-binding protein LIN41/TRIM71 regulates these processes by silencing only four target mRNAs. Thus, by silencing LIN41, let-7 activates LIN-29a and MAB-10 (an early growth response-type transcription factor and its NAB1/2-orthologous cofactor, respectively) to terminate progenitor cell self-renewal and to promote vulval integrity. By contrast, let-7 promotes development of the male sexual organ by up-regulating DMD-3 and MAB-3, two Doublesex/MAB-3 domain-containing transcription factors. Our results provide mechanistic insight into how a linear chain of post-transcriptional regulators diverges in the control of a small set of transcriptional regulators to achieve a coordinated J/A transition.

Timing mechanism of sexually dimorphic nervous system differentiation.

The molecular mechanisms that control the timing of sexual differentiation in the brain are poorly understood. We found that the timing of sexually dimorphic differentiation of postmitotic, sex-shared neurons in the nervous system of the Caenorhabditis elegans male is controlled by the temporally regulated miRNA let-7 and its target lin-41, a translational regulator. lin-41 acts through lin-29a, an isoform of a conserved Zn finger transcription factor, expressed in a subset of sex-shared neurons only in the male. Ectopic lin-29a is sufficient to impose male-specific features at earlier stages of development and in the opposite sex. The temporal, sexual and spatial specificity of lin-29a expression is controlled intersectionally through the lin-28/let-7/lin-41 heterochronic pathway, sex chromosome configuration and neuron-type-specific terminal selector transcription factors. Two Doublesex-like transcription factors represent additional sex- and neuron-type specific targets of LIN-41 and are regulated in a similar intersectional manner.

Evolutionary plasticity of the NHL domain underlies distinct solutions to RNA recognition.

RNA-binding proteins regulate all aspects of RNA metabolism. Their association with RNA is mediated by RNA-binding domains, of which many remain uncharacterized. A recently reported example is the NHL domain, found in prominent regulators of cellular plasticity like the C. elegans LIN-41. Here we employ an integrative approach to dissect the RNA specificity of LIN-41. Using computational analysis, structural biology, and in vivo studies in worms and human cells, we find that a positively charged pocket, specific to the NHL domain of LIN-41 and its homologs (collectively LIN41), recognizes a stem-loop RNA element, whose shape determines the binding specificity. Surprisingly, the mechanism of RNA recognition by LIN41 is drastically different from that of its more distant relative, the fly Brat. Our phylogenetic analysis suggests that this reflects a rapid evolution of the domain, presenting an interesting example of a conserved protein fold that acquired completely different solutions to RNA recognition.

The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan.

In Saccharomyces cerevisiae, deletion of large ribosomal subunit protein-encoding genes increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the messenger RNA and protein abundance, ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes, some encoding ribosomal proteins, that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations, including ribosomal protein gene deletions, calorie restriction, and rapamycin treatment, and provide an explanation for its role in longevity and stress response.The transcription factor Gcn4 is known to regulate yeast amino acid synthesis. Here, the authors show that Gcn4 also acts as a repressor of protein biosynthesis in a range of conditions that enhance yeast lifespan, such as ribosomal protein knockout, calorie restriction or mTOR inhibition.