The interaction between RPAP3 and TRBP reveals a possible involvement of the HSP90/R2TP chaperone complex in the regulation of miRNA activity.

MicroRNAs silence mRNAs by guiding the RISC complex. RISC assembly occurs following cleavage of pre-miRNAs by Dicer, assisted by TRBP or PACT, and the transfer of miRNAs to AGO proteins. The R2TP complex is an HSP90 co-chaperone involved in the assembly of ribonucleoprotein particles. Here, we show that the R2TP component RPAP3 binds TRBP but not PACT. The RPAP3-TPR1 domain interacts with the TRBP-dsRBD3, and the 1.5 Å resolution crystal structure of this complex identifies key residues involved in the interaction. Remarkably, binding of TRBP to RPAP3 or Dicer is mutually exclusive. Additionally, we found that AGO(1/2), TRBP and Dicer are all sensitive to HSP90 inhibition, and that TRBP sensitivity is increased in the absence of RPAP3. Finally, RPAP3 seems to impede miRNA activity, raising the possibility that the R2TP chaperone might sequester TRBP to regulate the miRNA pathway.

Synergistic defects in pre-rRNA processing from mutations in the U3-specific protein Rrp9 and U3 snoRNA.

U3 snoRNA and the associated Rrp9/U3-55K protein are essential for 18S rRNA production by the SSU-processome complex. U3 and Rrp9 are required for early pre-rRNA cleavages at sites A0, A1 and A2, but the mechanism remains unclear. Substitution of Arg 289 in Rrp9 to Ala (R289A) specifically reduced cleavage at sites A1 and A2. Surprisingly, R289 is located on the surface of the Rrp9 ß-propeller structure opposite to U3 snoRNA. To understand this, we first characterized the protein-protein interaction network of Rrp9 within the SSU-processome. This identified a direct interaction between the Rrp9 ß-propeller domain and Rrp36, the strength of which was reduced by the R289A substitution, implicating this interaction in the observed processing phenotype. The Rrp9 R289A mutation also showed strong synergistic negative interactions with mutations in U3 that destabilize the U3/pre-rRNA base-pair interactions or reduce the length of their linking segments. We propose that the Rrp9 ß-propeller and U3/pre-rRNA binding cooperate in the structure or stability of the SSU-processome. Additionally, our analysis of U3 variants gave insights into the function of individual segments of the 5'-terminal 72-nt sequence of U3. We interpret these data in the light of recently reported SSU-processome structures.

Identification of differentially expressed non-coding RNAs in embryonic stem cell neural differentiation.

Protein-coding genes, guiding differentiation of ES cells into neural cells, have extensively been studied in the past. However, for the class of ncRNAs only the involvement of some specific microRNAs (miRNAs) has been described. Thus, to characterize the entire small non-coding RNA (ncRNA) transcriptome, involved in the differentiation of mouse ES cells into neural cells, we have generated three specialized ribonucleo-protein particle (RNP)-derived cDNA libraries, i.e. from pluripotent ES cells, neural progenitors and differentiated neural cells, respectively. By high-throughput sequencing and transcriptional profiling we identified several novel miRNAs to be involved in ES cell differentiation, as well as seven small nucleolar RNAs. In addition, expression of 7SL, 7SK and vault-2 RNAs was significantly up-regulated during ES cell differentiation. About half of ncRNA sequences from the three cDNA libraries mapped to intergenic or intragenic regions, designated as interRNAs and intraRNAs, respectively. Thereby, novel ncRNA candidates exhibited a predominant size of 18-30 nt, thus resembling miRNA species, but, with few exceptions, lacking canonical miRNA features. Additionally, these novel intraRNAs and interRNAs were not only found to be differentially expressed in stem-cell derivatives, but also in primary cultures of hippocampal neurons and astrocytes, strengthening their potential function in neural ES cell differentiation.

[Beyond usual functions of snoRNAs]. / Les petits ARN nucléolaires nous surprennent encore !

Small nucleolar RNAs or snoRNAs, principally implicated in post-transcriptional chemical modification of other RNAs, were among the first non-coding RNA identified, together with ribosomal and transfer RNA. Lately, snoRNA have been involved in various unexpected functions, which renewed researcher's interest for these molecules. SnoRNA processing into smaller functional RNA species (sdRNA for snoRNA-derived RNA) or into miRNA (sno-miR), snoRNA mediated regulation of messenger RNA alternative splicing or snoRNA links to human disorders, including cancers, are some of the topics developed in this review.

Satellite cell loss and impaired muscle regeneration in selenoprotein N deficiency.

Selenoprotein N (SelN) deficiency causes a group of inherited neuromuscular disorders termed SEPN1-related myopathies (SEPN1-RM). Although the function of SelN remains unknown, recent data demonstrated that it is dispensable for mouse embryogenesis and suggested its involvement in the regulation of ryanodine receptors and/or cellular redox homeostasis. Here, we investigate the role of SelN in satellite cell (SC) function and muscle regeneration, using the Sepn1(-/-) mouse model. Following cardiotoxin-induced injury, SelN expression was strongly up-regulated in wild-type muscles and, for the first time, we detected its endogenous expression in a subset of mononucleated cells by immunohistochemistry. We show that SelN deficiency results in a reduced basal SC pool in adult skeletal muscles and in an imperfect muscle restoration following a single injury. A dramatic depletion of the SC pool was detected after the first round of degeneration and regeneration that totally prevented subsequent regeneration of Sepn1(-/-) muscles. We demonstrate that SelN deficiency affects SC dynamics on isolated single fibres and increases the proliferation of Sepn1(-/-) muscle precursors in vivo and in vitro. Most importantly, exhaustion of the SC population was specifically identified in muscle biopsies from patients with mutations in the SEPN1 gene. In conclusion, we describe for the first time a major physiological function of SelN in skeletal muscles, as a key regulator of SC function, which likely plays a central role in the pathophysiological mechanism leading to SEPN1-RM.

Direct cloning of double-stranded RNAs from RNase protection analysis reveals processing patterns of C/D box snoRNAs and provides evidence for widespread antisense transcript expression.

We describe a new method that allows cloning of double-stranded RNAs (dsRNAs) that are generated in RNase protection experiments. We demonstrate that the mouse C/D box snoRNA MBII-85 (SNORD116) is processed into at least five shorter RNAs using processing sites near known functional elements of C/D box snoRNAs. Surprisingly, the majority of cloned RNAs from RNase protection experiments were derived from endogenous cellular RNA, indicating widespread antisense expression. The cloned dsRNAs could be mapped to genome areas that show RNA expression on both DNA strands and partially overlapped with experimentally determined argonaute-binding sites. The data suggest a conserved processing pattern for some C/D box snoRNAs and abundant expression of longer, non-coding RNAs in the cell that can potentially form dsRNAs.

RNPomics: defining the ncRNA transcriptome by cDNA library generation from ribonucleo-protein particles.

Up to 450,000 non-coding RNAs (ncRNAs) have been predicted to be transcribed from the human genome. However, it still has to be elucidated which of these transcripts represent functional ncRNAs. Since all functional ncRNAs in Eukarya form ribonucleo-protein particles (RNPs), we generated specialized cDNA libraries from size-fractionated RNPs and validated the presence of selected ncRNAs within RNPs by glycerol gradient centrifugation. As a proof of concept, we applied the RNP method to human Hela cells or total mouse brain, and subjected cDNA libraries, generated from the two model systems, to deep-sequencing. Bioinformatical analysis of cDNA sequences revealed several hundred ncRNP candidates. Thereby, ncRNAs candidates were mainly located in intergenic as well as intronic regions of the genome, with a significant overrepresentation of intron-derived ncRNA sequences. Additionally, a number of ncRNAs mapped to repetitive sequences. Thus, our RNP approach provides an efficient way to identify new functional small ncRNA candidates, involved in RNP formation.

Stem-Loop qRT-PCR-Based Quantification of miRNAs.

Stem-loop qRT-PCR is one of the most commonly used real-time PCR approach to quantify small non-coding RNAs such as microRNAs. The quantification method is divided in two steps. First, RNA is reverse transcribed using a specific stem-loop primer, and the resulting RT product is subsequently used as a template for quantitative real-time PCR. This fast and simple method provides quantitative data with high sensitivity and specificity to study miRNAs and their functions.

Gene Reporter Assays to Study miRNA Function.

This chapter describes one of the most reliable quantitative assays to test the silencing of a possible target gene by a specific miRNA using a luciferase reporter gene. The experimental procedure first consists in cloning both the wild-type and mutated forms of the 3'UTR of the miRNA-predicted mRNA target downstream of a firefly luciferase reporter. Next, each construct is co-transfected together with the miRNA into HeLa cells, and the reporter expression is monitored. Changes in luciferase levels will indicate whether or not an miRNA can bind to the UTR and regulate its expression.

RNA Precipitation.

Precipitation is a critical step to recover RNA of high purity. This chapter describes the principles of alcoholic precipitation as well as a standard, basic protocol with key advices to observe, but numerous variations on the theme are discussed. Indeed, several important parameters, such as the choice of salt, alcohol, or carrier, have to be considered to improve the efficiency of precipitation and the yield of RNA recovery.

Regulatory Non-Coding RNAs: An Overview.

The discovery of new classes of non-coding RNAs has always been preceded or accompanied by technological breakthroughs, and these outstanding progresses in transcriptomics approaches enabled to regularly add new members to the list. From the first detection of tRNAs, through the revolution of miRNAs discovery, to the recent identification of eRNAs or the identification of new functions for already known ncRNAs, this introductive review provides a very concise historical and functional overview of most prominent small regulatory non-coding RNA families.

Identification of oxytocin-related lncRNAs and assessment of their expression in breast cancer.

Oxytocin is a neuropeptide released by the central nervous system. A number of studies have demonstrated the role of this neuropeptide in the pathogenesis of breast cancer. In the present project, we have identified mRNA coding genes and long non-coding RNAs (lncRNAs) that are associated with this pathway through an in-silico strategy, and measured their expression in a cohort of Iranian females affected with this type of malignancy. Expression levels of OXTR, FOS, ITPR1, RCAN1, CAMK2D, CACNA2D and lnc_ZFP161 were significantly down-regulated in breast cancer tissues compared with nearby non-cancerous tissues. On the other hand, expression of lnc_MTX2 was higher in breast cancer tissues compared with controls. Expression of lnc_TNS1 and lnc_FOXF1 were not different between these two kinds of samples. Expression of CACNA2D was associated with mitotic rate and PR status (P values = 3.02E-02 and 2.53E-02, respectively). Expression of other oxytocin-related genes was not associated with clinicopathological parameters. FOS and ITPR1 had the highest AUC value among the oxytocin-related genes. Combination of expression profiles of all oxytocin-related genes increased the AUC value to 0.75. However, the combinatorial sensitivity and specificity values were lower than some individual genes. In the breast cancer tissues, the most robust correlations have been detected between lnc_ZFP161/ lnc_FOXF1, CAMK2D/ lnc_ZFP161 and CAMK2D / lnc_FOXF1 (r = 0.86, 0.71 and 0.64 respectively). In the non-cancerous tissues, the strongest correlation was detected between lnc_FOXF1/lnc_MTX2 and lnc_ZFP161/CAMK2D respectively (r = 0.78 and 0.65). Taken together, oxytocin-associated genes have been dysregulated in breast cancer tissues. Moreover, the correlation ratio between these genes is connected with the existence of cancer.

Compartmentalization and regulation of mitochondrial function by methionine sulfoxide reductases in yeast.

Elevated levels of reactive oxygen species can damage proteins. Sulfur-containing amino acid residues, cysteine and methionine, are particularly susceptible to such damage. Various enzymes evolved to protect proteins or repair oxidized residues, including methionine sulfoxide reductases MsrA and MsrB, which reduce methionine (S)-sulfoxide (Met-SO) and methionine (R)-sulfoxide (Met-RO) residues, respectively, back to methionine. Here, we show that MsrA and MsrB are involved in the regulation of mitochondrial function. Saccharomyces cerevisiae mutant cells lacking MsrA, MsrB, or both proteins had normal levels of mitochondria but lower levels of cytochrome c and fewer respiration-competent mitochondria. The growth of single MsrA or MsrB mutants on respiratory carbon sources was inhibited, and that of the double mutant was severely compromised, indicating impairment of mitochondrial function. Although MsrA and MsrB are thought to have similar roles in oxidative protein repair each targeting a diastereomer of methionine sulfoxide, their deletion resulted in different phenotypes. GFP fusions of MsrA and MsrB showed different localization patterns and primarily localized to cytoplasm and mitochondria, respectively. This finding agreed with compartment-specific enrichment of MsrA and MsrB activities. These results show that oxidative stress contributes to mitochondrial dysfunction through oxidation of methionine residues in proteins located in different cellular compartments.

Selenoprotein function and muscle disease.

The crucial role of the trace element selenium in livestock and human health, in particular in striated muscle function, has been well established but the underlying molecular mechanisms remain poorly understood. Over the last decade, identification of the full repertoire of selenium-containing proteins has opened the way towards a better characterization of these processes. Two selenoproteins have mainly been investigated in muscle, namely SelW and SelN. Here we address their involvement in muscle development and maintenance, through the characterization of various cellular or animal models. In particular, mutations in the SEPN1 gene encoding selenoprotein N (SelN) cause a group of neuromuscular disorders now referred to as SEPN1-related myopathy. Recent findings on the functional consequences of these mutations suggest an important contribution of SelN to the regulation of oxidative stress and calcium homeostasis. Importantly, the conclusions of these experiments have opened new avenues of investigations that provide grounds for the development of therapeutic approaches.

Small ncRNA transcriptome analysis from Aspergillus fumigatus suggests a novel mechanism for regulation of protein synthesis.

Small non-protein-coding RNAs (ncRNAs) have systematically been studied in various model organisms from Escherichia coli to Homo sapiens. Here, we analyse the small ncRNA transcriptome from the pathogenic filamentous fungus Aspergillus fumigatus. To that aim, we experimentally screened for ncRNAs, expressed under various growth conditions or during specific developmental stages, by generating a specialized cDNA library from size-selected small RNA species. Our screen revealed 30 novel ncRNA candidates from known ncRNA classes such as small nuclear RNAs (snRNAs) and C/D box-type small nucleolar RNAs (C/D box snoRNAs). Additionally, several candidates for H/ACA box snoRNAs could be predicted by a bioinformatical screen. We also identified 15 candidates for ncRNAs, which could not be assigned to any known ncRNA class. Some of these ncRNA species are developmentally regulated implying a possible novel function in A. fumigatus development. Surprisingly, in addition to full-length tRNAs, we also identified 5'- or 3'-halves of tRNAs, only, which are likely generated by tRNA cleavage within the anti-codon loop. We show that conidiation induces tRNA cleavage resulting in tRNA depletion within conidia. Since conidia represent the resting state of A. fumigatus we propose that conidial tRNA depletion might be a novel mechanism to down-regulate protein synthesis in a filamentous fungus.

Selenoprotein N is dynamically expressed during mouse development and detected early in muscle precursors.

BACKGROUND: In humans, mutations in the SEPN1 gene, encoding selenoprotein N (SelN), are involved in early onset recessive neuromuscular disorders, referred to as SEPN1-related-myopathies. The mechanisms behind these pathologies are poorly understood since the function of SelN remains elusive. However, previous results obtained in humans and more recently in zebrafish pointed to a potential role for SelN during embryogenesis. Using qRT-PCR, Western blot and whole mount in situ hybridization, we characterized in detail the spatio-temporal expression pattern of the murine Sepn1 gene during development, focusing particularly on skeletal muscles. RESULTS: In whole embryos, Sepn1 transcripts were detected as early as E5.5, with expression levels peaking at E12.5, and then strongly decreasing until birth. In isolated tissues, only mild transcriptional variations were observed during development, whereas a striking reduction of the protein expression was detected during the perinatal period. Furthermore, we demonstrated that Sepn1 is expressed early in somites and restricted to the myotome, the sub-ectodermal mesenchyme and the dorsal root ganglia at mid-gestation stages. Interestingly, Sepn1 deficiency did not alter somitogenesis in embryos, suggesting that SelN is dispensable for these processes in mouse. CONCLUSION: We characterized for the first time the expression pattern of Sepn1 during mammalian embryogenesis and we demonstrated that its differential expression is most likely dependent on major post-transcriptional regulations. Overall, our data strongly suggest a potential role for selenoprotein N from mid-gestation stages to the perinatal period. Interestingly, its specific expression pattern could be related to the current hypothesis that selenoprotein N may regulate the activity of the ryanodine receptors.

SnoRNAs and the emerging class of sdRNAs: Multifaceted players in oncogenesis.

Small nucleolar RNAs are generally involved in the modification of target ribosomal RNAs. Other possible functions recently emerged with the discovery of the novel class of snoRNA-derived RNAs or sdRNAs. Since then, additional data has revealed the involvement of both snoRNAs and sdRNAs in tumorigenesis. After briefly introducing snoRNA families and functions, this mini-review summarises recently acquired knowledge on snoRNA-related mechanisms associated with cancer. Despite the rapid increase in the number of studies, exactly how snoRNAs lead to cancer remains unclear. However, exciting new research is paving the way for future diagnostic or therapeutic strategies.