A rapid protocol for ribosome profiling of low input samples.

Ribosome profiling provides quantitative, comprehensive, and high-resolution snapshots of cellular translation by the high-throughput sequencing of short mRNA fragments that are protected by ribosomes from nucleolytic digestion. While the overall principle is simple, the workflow of ribosome profiling experiments is complex and challenging, and typically requires large amounts of sample, limiting its broad applicability. Here, we present a new protocol for ultra-rapid ribosome profiling from low-input samples. It features a robust strategy for sequencing library preparation within one day that employs solid phase purification of reaction intermediates, allowing to reduce the input to as little as 0.1 pmol of ∼30 nt RNA fragments. Hence, it is particularly suited for the analyses of small samples or targeted ribosome profiling. Its high sensitivity and its ease of implementation will foster the generation of higher quality data from small samples, which opens new opportunities in applying ribosome profiling.

Identification of novel targets of miR-622 in hepatocellular carcinoma reveals common regulation of cooperating genes and outlines the oncogenic role of zinc finger CCHC-type containing 11.

The poor prognosis of advanced hepatocellular carcinoma (HCC) is driven by diverse features including dysregulated microRNAs inducing drug resistance and stemness. Lin-28 homolog A (LIN28A) and its partner zinc finger CCHC-type containing 11 (ZCCHC11) cooperate in binding, oligouridylation and subsequent degradation of tumorsuppressive let-7 precursor microRNAs. Functionally, activation of LIN28A was recently shown to promote stemness and chemoresistance in HCC. However, the expression and regulation of LIN28A in HCC had been unclear. Moreover, the expression, regulation and function of ZCCHC11 in liver cancer remained elusive. In contrast to "one-microRNA-one-target" interactions, we identified common binding sites for miR-622 in both LIN28A and ZCCHC11, suggesting miR-622 to function as a superior pathway regulator. Applying comprehensive microRNA database screening, human hepatocytes and HCC cell lines, patient-derived tissue samples as well as "The Cancer Genome Atlas" (TCGA) patient cohorts, we demonstrated that loss of tumorsuppressive miR-622 mediates derepression and overexpression of LIN28A in HCC. Moreover, the cooperator of LIN28A, ZCCHC11, was newly identified as a prognostic and therapeutic target of miR-622 in liver cancer. Together, identification of novel miR-622 target genes revealed common regulation of cooperating genes and outlines the previously unknown oncogenic role of ZCCHC11 in liver cancer.

Prevention of dsRNA-induced interferon signaling by AGO1x is linked to breast cancer cell proliferation.

Translational readthrough, i.e., elongation of polypeptide chains beyond the stop codon, was initially reported for viral RNA, but later found also on eukaryotic transcripts, resulting in proteome diversification and protein-level modulation. Here, we report that AGO1x, an evolutionarily conserved translational readthrough isoform of Argonaute 1, is generated in highly proliferative breast cancer cells, where it curbs accumulation of double-stranded RNAs (dsRNAs) and consequent induction of interferon responses and apoptosis. In contrast to other mammalian Argonaute protein family members with primarily cytoplasmic functions, AGO1x exhibits nuclear localization in the vicinity of nucleoli. We identify AGO1x interaction with the polyribonucleotide nucleotidyltransferase 1 (PNPT1) and show that the depletion of this protein further augments dsRNA accumulation. Our study thus uncovers a novel function of an Argonaute protein in buffering the endogenous dsRNA-induced interferon responses, different than the canonical function of AGO proteins in the miRNA effector pathway. As AGO1x expression is tightly linked to breast cancer cell proliferation, our study thus suggests a new direction for limiting tumor growth.

The Long Non-coding RNA lnc-31 Interacts with Rock1 mRNA and Mediates Its YB-1-Dependent Translation.

Cytoplasmic long non-coding RNAs have been shown to act at many different levels to control post-transcriptional gene expression, although their role in translational control is poorly understood. Here, we show that lnc-31, a non-coding RNA required for myoblast proliferation, promotes ROCK1 protein synthesis by stabilizing its translational activator, YB-1. We find that lnc-31 binds to the Rock1 mRNA as well as to the YB-1 protein and that translational activation requires physical interaction between the two RNA species. These results suggest a localized effect of YB-1 stabilization on the Rock1 mRNA. ROCK1 upregulation by lnc-31, in proliferative conditions, correlates well with the differentiation-repressing activity of ROCK1. We also show that, upon induction of differentiation, the downregulation of lnc-31, in conjunction with miR-152 targeting of Rock1, establishes a regulatory loop that reinforces ROCK1 repression and promotes myogenesis.

Phosphorylation of Argonaute proteins affects mRNA binding and is essential for microRNA-guided gene silencing in vivo.

Argonaute proteins associate with microRNAs and are key components of gene silencing pathways. With such a pivotal role, these proteins represent ideal targets for regulatory post-translational modifications. Using quantitative mass spectrometry, we find that a C-terminal serine/threonine cluster is phosphorylated at five different residues in human and Caenorhabditis elegans In human, hyper-phosphorylation does not affect microRNA binding, localization, or cleavage activity of Ago2. However, mRNA binding is strongly affected. Strikingly, on Ago2 mutants that cannot bind microRNAs or mRNAs, the cluster remains unphosphorylated indicating a role at late stages of gene silencing. In C. elegans, the phosphorylation of the conserved cluster of ALG-1 is essential for microRNA function in vivo Furthermore, a single point mutation within the cluster is sufficient to phenocopy the loss of its complete phosphorylation. Interestingly, this mutant retains its capacity to produce and bind microRNAs and represses expression when artificially tethered to an mRNA Altogether, our data suggest that the phosphorylation state of the serine/threonine cluster is important for Argonaute-mRNA interactions.

Peptide-Based Inhibition of miRNA-Guided Gene Silencing.

MicroRNAs (miRNAs) are a large class of small noncoding RNAs that regulate the expression of distinct target mRNAs. miRNAs are incorporated into Argonaute (AGO) proteins and guide them to their target mRNAs. Subsequently, AGO proteins recruit a member of the glycine-tryptophan-rich (GW) protein family by direct protein-protein interaction. GW proteins coordinate all downstream processes leading to robust and efficient gene silencing. A short peptide of GW proteins comprising the AGO interaction motif can be used to biochemically isolate endogenous AGO protein complexes. Furthermore, within a cell such a peptide competes with endogenous GW proteins for AGO binding and thus can be used as potent inhibitor of the miRNA pathway. Here, we describe a method that utilizes a GW-based polypeptide (T6B-assay) to validate miRNA-mRNA interactions in tissue culture systems.

Importin-ß facilitates nuclear import of human GW proteins and balances cytoplasmic gene silencing protein levels.

MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-ß pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago - TNRC6 levels.