What goes up must come down: off switches for regulatory RNAs.

Small RNAs base pair with and regulate mRNA translation and stability. For both bacterial small regulatory RNAs and eukaryotic microRNAs, association with partner proteins is critical for the stability and function of the regulatory RNAs. We review the mechanisms for degradation of these RNAs: displacement of the regulatory RNA from its protein partner (in bacteria) or destruction of the protein and its associated microRNAs (in eukaryotes).

Catalytic residues of microRNA Argonautes play a modest role in microRNA star strand destabilization in C. elegans.

Many microRNA (miRNA)-guided Argonaute proteins can cleave RNA ('slicing'), even though miRNA-mediated target repression is generally cleavage-independent. Here we use Caenorhabditis elegans to examine the role of catalytic residues of miRNA Argonautes in organismal development. In contrast to previous work, mutations in presumed catalytic residues did not interfere with development when introduced by CRISPR.

The E3 ligase Poe promotes Pericentrin degradation.

Centrosomes are essential parts of diverse cellular processes, and precise regulation of the levels of their constituent proteins is critical for their function. One such protein is Pericentrin (PCNT) in humans and Pericentrin-like protein (PLP) in . Increased PCNT expression and its protein accumulation are linked to clinical conditions including cancer, mental disorders, and ciliopathies.

Recent advances in understanding microRNA function and regulation in C. elegans.

MicroRNAs (miRNAs) were first discovered in C. elegans as essential post-transcriptional regulators of gene expression. Since their initial discovery, miRNAs have been implicated in numerous areas of physiology and disease in all animals examined.

The catalytic activity of microRNA Argonautes plays a modest role in microRNA star strand destabilization in .

Many Argonaute proteins can cleave RNA ("slicing") as part of the microRNA-induced silencing complex (miRISC), even though miRNA-mediated target repression is generally independent of target cleavage. Here we use genome editing in to examine the role of miRNA-guided slicing in organismal development. In contrast to previous work, slicing-inactivating mutations did not interfere with normal development when introduced by CRISPR.

The developmentally timed decay of an essential microRNA family is seed-sequence dependent.

MicroRNA (miRNA) abundance is tightly controlled by regulation of biogenesis and decay. Here, we show that the mir-35 miRNA family undergoes selective decay at the transition from embryonic to larval development in C. elegans.

Screening by deep sequencing reveals mediators of microRNA tailing in C. elegans.

microRNAs are frequently modified by addition of untemplated nucleotides to the 3' end, but the role of this tailing is often unclear. Here we characterize the prevalence and functional consequences of microRNA tailing in vivo, using Caenorhabditis elegans. MicroRNA tailing in C.

In vivo CRISPR screening for phenotypic targets of the family in .

Identifying miRNA target genes is difficult, and delineating which targets are the most biologically important is even more difficult. We devised a novel strategy to test the phenotypic impact of individual microRNA-target interactions by disrupting each predicted miRNA-binding site by CRISPR-Cas9 genome editing in We developed a multiplexed negative selection screening approach in which edited loci are deep sequenced, and candidate sites are prioritized based on apparent selection pressure against mutations that disrupt miRNA binding. Importantly, our screen was conducted in vivo on mutant animals, allowing us to interrogate organism-level phenotypes.

CRISPR screening strategies for microRNA target identification.

Identifying microRNA (miRNA) target genes remains a major challenge in understanding the roles miRNAs play in gene regulation. Furthermore, understanding which miRNA-target interactions are the most biologically important is even more difficult. We present CRISPR-based strategies to identify essential miRNA binding sites.

The Family Links Maternal Germline Sex to Embryonic Viability in .

The germline sex determination pathway in determines whether germ cells develop as oocytes or sperm, with no previously known effect on viability. The family of microRNAs are expressed in the germline and embryo and are essential for both viability and normal hermaphroditic sex determination, preventing aberrant male gene expression in XX hermaphrodite embryos. Here we show that combining feminizing mutations with partial loss of function of the family results in enhanced penetrance embryonic lethality that preferentially kills XO animals.

The TRIM-NHL protein NHL-2 is a co-factor in the nuclear and somatic RNAi pathways in . e.

Proper regulation of germline gene expression is essential for fertility and maintaining species integrity. In the germline, a diverse repertoire of regulatory pathways promote the expression of endogenous germline genes and limit the expression of deleterious transcripts to maintain genome homeostasis. Here we show that the conserved TRIM-NHL protein, NHL-2, plays an essential role in the germline, modulating germline chromatin and meiotic chromosome organization.

Maternal effects of microRNAs in early embryogenesis.

The window of embryonic development after fertilization but prior to the beginning of transcription from the zygotic genome is a period that relies heavily on post-transcriptional regulation of gene expression. MicroRNAs constitute one of the predominant mechanisms of post-transcriptional gene regulation, yet their biological function and molecular mechanism of action during this developmental window is poorly understood. Our recent findings demonstrate that the maternal contribution of mir-35 family members contributes to zygotic developmental decisions (sex determination) in C.

A microRNA family exerts maternal control on sex determination in .

Gene expression in early animal embryogenesis is in large part controlled post-transcriptionally. Maternally contributed microRNAs may therefore play important roles in early development. We elucidated a major biological role of the nematode family of maternally contributed essential microRNAs.

miRNAs cooperate in apoptosis regulation during development.

Programmed cell death occurs in a highly reproducible manner during development. We demonstrate that, during embryogenesis, miR-35 and miR-58 family microRNAs (miRNAs) cooperate to prevent the precocious death of mothers of cells programmed to die by repressing the gene , which encodes a proapoptotic BH3-only protein. In addition, we present evidence that repression of is dependent on binding sites for miR-35 and miR-58 family miRNAs within the 3' untranslated region (UTR), which affect both mRNA copy number and translation.

Correction: GW182-Free microRNA Silencing Complex Controls Post-transcriptional Gene Expression during Caenorhabditis elegans Embryogenesis.

[This corrects the article DOI: 10.1371/journal.pgen.

GW182-Free microRNA Silencing Complex Controls Post-transcriptional Gene Expression during Caenorhabditis elegans Embryogenesis.

MicroRNAs and Argonaute form the microRNA induced silencing complex or miRISC that recruits GW182, causing mRNA degradation and/or translational repression. Despite the clear conservation and molecular significance, it is unknown if miRISC-GW182 interaction is essential for gene silencing during animal development. Using Caenorhabditis elegans to explore this question, we examined the relationship and effect on gene silencing between the GW182 orthologs, AIN-1 and AIN-2, and the microRNA-specific Argonaute, ALG-1.

In vivo RNAi screening identifies a mechanism of sorafenib resistance in liver cancer.

In solid tumors, resistance to therapy inevitably develops upon treatment with cytotoxic drugs or molecularly targeted therapies. Here, we describe a system that enables pooled shRNA screening directly in mouse hepatocellular carcinomas (HCC) in vivo to identify genes likely to be involved in therapy resistance. Using a focused shRNA library targeting genes located within focal genomic amplifications of human HCC, we screened for genes whose inhibition increased the therapeutic efficacy of the multikinase inhibitor sorafenib.

The embryonic mir-35 family of microRNAs promotes multiple aspects of fecundity in Caenorhabditis elegans.

MicroRNAs guide many aspects of development in all metazoan species. Frequently, microRNAs are expressed during a specific developmental stage to perform a temporally defined function. The C.

A pipeline for the generation of shRNA transgenic mice.

RNA interference (RNAi) is an extremely effective tool for studying gene function in almost all metazoan and eukaryotic model systems. RNAi in mice, through the expression of short hairpin RNAs (shRNAs), offers something not easily achieved with traditional genetic approaches-inducible and reversible gene silencing. However, technical variability associated with the production of shRNA transgenic strains has so far limited their widespread use.

Reversible suppression of an essential gene in adult mice using transgenic RNA interference.

RNAi has revolutionized loss-of-function genetics by enabling sequence-specific suppression of virtually any gene. Furthermore, tetracycline response elements (TRE) can drive expression of short hairpin RNAs (shRNAs) for inducible and reversible target gene suppression. Here, we demonstrate the feasibility of transgenic inducible RNAi for suppression of essential genes.

Functional identification of optimized RNAi triggers using a massively parallel sensor assay.

Short hairpin RNAs (shRNAs) provide powerful experimental tools by enabling stable and regulated gene silencing through programming of endogenous microRNA pathways. Since requirements for efficient shRNA biogenesis and target suppression are largely unknown, many predicted shRNAs fail to efficiently suppress their target. To overcome this barrier, we developed a "Sensor assay" that enables the biological identification of effective shRNAs at large scale.

Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi.

Short hairpin RNAs (shRNAs) are versatile tools for analyzing loss-of-function phenotypes in vitro and in vivo. However, their use for studying genes involved in proliferation and survival, which are potential therapeutic targets in cancer and other diseases, is confounded by the strong selective advantage of cells in which shRNA expression is inefficient. We therefore developed a toolkit that combines Tet-regulated miR30-shRNA technology, robust transactivator expression and two fluorescent reporters to track and isolate cells with potent target knockdown.

Genome-wide RNA-mediated interference screen identifies miR-19 targets in Notch-induced T-cell acute lymphoblastic leukaemia.

MicroRNAs (miRNAs) have emerged as novel cancer genes. In particular, the miR-17-92 cluster, containing six individual miRNAs, is highly expressed in haematopoietic cancers and promotes lymphomagenesis in vivo. Clinical use of these findings hinges on isolating the oncogenic activity within the 17-92 cluster and defining its relevant target genes.