Nt RNA Research Articles

Identification of plasma microRNAs as a biomarker of sporadic Amyotrophic Lateral Sclerosis.

BackgroundAmyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease, which leads to the loss of upper and lower motor neurons, with a currently unknown etiology. Specific biomarkers could help in early detection and diagnosis, and could also act as indicators of disease progression and therapy effectiveness. MicroRNAs (miRNAs) are small (18–25 nucleotides), single-stranded non-coding RNA molecules that play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression, and are essential for nervous system development. Many of the genes associated with genetic ALS have pathological biological pathways related to RNA metabolism, and their pathogenesis may be affecting the maturing processes of miRNA.ResultsWe compared miRNA from the plasma of sALS patients and healthy controls using two cohorts; a discovery cohort analyzed with microarray (16 sALS patients and ten healthy controls) and a validation cohort confirmed with qPCR (48 sALS patients, 47 healthy controls and 30 disease controls). We measured the total amount of extracted RNA along with a spike-in control that ensured the quality of our quantification. A percentage of the 10–40 nt RNAs extracted from the total RNA showed a significant increase in ALS patients. There was a negative correlation between total RNA concentration and disease duration from onset to end point. Three of the miRNAs were up-regulated and six were down-regulated significantly in the discovery cohort. Since an internal control is required as a sample stability indicator of both the patients and controls in microarray analysis, we selected the miRNA showing the smallest dispersion and equivalency between the two groups’ mean value, and decided to use hsa-miR-4516. We found hsa-miR-4649-5p to be up-regulated, and hsa-miR-4299 to be down-regulated, where each was not influenced by clinical characteristics. EPHA4, a target gene linked to the nervous system which has also been reported to be a disease modifier of ALS, is the common and most notable target gene of hsa-miR-4649-5p and hsa-miR-4299.ConclusionWe have shown the relationship circulating plasma miRNA has with both healthy controls and diseased patients. Hsa-miR-4649-5p and hsa-miR-4299 have the potential to be ALS diagnosis biomarkers.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0161-7) contains supplementary material, which is available to authorized users.

Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis.

In Arabidopsis thaliana, abundant 24 nucleotide small interfering RNAs (24 nt siRNA) guide the cytosine methylation and silencing of transposons and a subset of genes. 24 nt siRNA biogenesis requires nuclear RNA polymerase IV (Pol IV), RNA-dependent RNA polymerase 2 (RDR2) and DICER-like 3 (DCL3). However, siRNA precursors are mostly undefined. We identified Pol IV and RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl3 mutants and are diced into 24 nt RNAs by DCL3 in vitro. P4R2 RNAs are mostly 26-45 nt and initiate with a purine adjacent to a pyrimidine, characteristics shared by Pol IV transcripts generated in vitro. RDR2 terminal transferase activity, also demonstrated in vitro, may account for occasional non-templated nucleotides at P4R2 RNA 3' termini. The 24 nt siRNAs primarily correspond to the 5' or 3' ends of P4R2 RNAs, suggesting a model whereby siRNAs are generated from either end of P4R2 duplexes by single dicing events.

Conserved Motifs within Hepatitis C Virus Envelope (E2) RNA and Protein Independently Inhibit T Cell Activation.

T cell receptor (TCR) signaling is required for T-cell activation, proliferation, differentiation, and effector function. Hepatitis C virus (HCV) infection is associated with impaired T-cell function leading to persistent viremia, delayed and inconsistent antibody responses, and mild immune dysfunction. Although multiple factors appear to contribute to T-cell dysfunction, a role for HCV particles in this process has not been identified. Here, we show that incubation of primary human CD4+ and CD8+ T-cells with HCV RNA-containing serum, HCV-RNA containing extracellular vesicles (EVs), cell culture derived HCV particles (HCVcc) and HCV envelope pseudotyped retrovirus particles (HCVpp) inhibited TCR-mediated signaling. Since HCVpp’s contain only E1 and E2, we examined the effect of HCV E2 on TCR signaling pathways. HCV E2 expression recapitulated HCV particle-induced TCR inhibition. A highly conserved, 51 nucleotide (nt) RNA sequence was sufficient to inhibit TCR signaling. Cells expressing the HCV E2 coding RNA contained a short, virus-derived RNA predicted to be a Dicer substrate, which targeted a phosphatase involved in Src-kinase signaling (PTPRE). T-cells and hepatocytes containing HCV E2 RNA had reduced PTPRE protein levels. Mutation of 6 nts abolished the predicted Dicer interactions and restored PTPRE expression and proximal TCR signaling. HCV RNA did not inhibit distal TCR signaling induced by PMA and Ionomycin; however, HCV E2 protein inhibited distal TCR signaling. This inhibition required lymphocyte-specific tyrosine kinase (Lck). Lck phosphorylated HCV E2 at a conserved tyrosine (Y613), and phospho-E2 inhibited nuclear translocation of NFAT. Mutation of Y613 restored distal TCR signaling, even in the context of HCVpps. Thus, HCV particles delivered viral RNA and E2 protein to T-cells, and these inhibited proximal and distal TCR signaling respectively. These effects of HCV particles likely aid in establishing infection and contribute to viral persistence.

RAID3 - An interleukin-6 receptor-binding aptamer with post-selective modification-resistant affinity

Aptamers are an emerging class of highly specific targeting ligands. They can be selected in vitro for a large variety of targets, ranging from small molecules to whole cells. Most aptamers selected are nucleic acid-based, allowing chemical synthesis and easy modification. Although their properties make them interesting drug candidates for a broad spectrum of applications and an interesting alternative to antibodies or fusion proteins, they are not yet broadly used. One major drawback of aptamers is their susceptibility to abundant serum nucleases, resulting in their fast degradation in biological fluids. Using modified nucleic acids has become a common strategy to overcome these disadvantages, greatly increasing their half-life under cell culture conditions or even in vivo. Whereas pre-selective modifications of the initial library for aptamer selection are relatively easy to obtain, post-selective modifications of already selected aptamers are still generally very labor-intensive and often compromise the aptamers ability to bind its target molecule.Here we report the selection, characterization and post-selective modification of a 34 nucleotide (nt) RNA aptamer for a non-dominant, novel target site (domain 3) of the interleukin-6 receptor (IL-6R). We performed structural analyses and investigated the affinity of the aptamer to the membrane-bound and soluble forms (sIL-6R) of the IL-6R. Further, we performed structural analyses of the aptamer in solution using small-angle X-ray scattering and determined its overall shape and oligomeric state. Post-selective exchange of all pyrimidines against their 2′-fluoro analogs increased the aptamers stability significantly without compromising its affinity for the target protein. The resulting modified aptamer could be shortened to its minimal binding motif without loss of affinity.

Abstract LB-296: Small RNA profiling of human testicular germ cell tumor tissue samples shows abundant tRNA fragments and global loss of piRNA

Abstract Small non-coding RNAs play essential roles in tumorigenesis. However, their role in testicular germ cell tumor (TGCT), the most common malignancy in young males in most western countries, is largely unknown. In particular, a comprehensive analysis of the small RNA composition, including microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and tRNA-derived small RNAs and their interactions, are lacking. tRNA-derived small RNAs have been shown to bind to the piRNA-associated protein HIWI2, indicating crosstalk between small RNA pathways. We have employed small RNA sequencing on 22 human TGCT samples from 5 histological subtypes, 3 carcinoma in situ, and 12 normal testis samples. The sequences from each sample group were analyzed according to sequence length, base composition, and their association with repeats, exons, tRNAs, piRNAs, and miRNAs. Most sequences 24-32 nucleotides in length, displayed piRNA characteristics. Expression analyses of the small RNA contigs demonstrated global loss of small RNAs in TGCT compared to normal testis. A large proportion of the 33-36 nt RNA sequences derives from mature tRNAs, predominantly from the 5′ end. We did not identify differences in the expression of 5′ tRNA halves between normal and cancer tissue samples; however, 3 shorter 5′ tRNA fragments showed differential expression. Although components of the same pathways might be involved in piRNA and tRNA-derived small RNAs biogenesis, expression differences in TGCT samples suggest independent regulation of key components in response to the carcinogenic process. Our results show subtype-specific microRNA expression, as well as a global loss of piRNAs in all TGCT histological subtypes when compared to normal testicular tissue. In addition, we document the presence of large amounts of tRNA-derived sequences in both TGCT and normal samples, which may provide new insight into the association between the tRNA fragments and piRNA biogenesis. These results may be valuable in the identification of new biomarkers for TGCT. Citation Format: Trine B. Rounge, Kari Furu, Rolf Skotheim, Trine B. Haugen, Espen Enerly, Tom Grotmol. Small RNA profiling of human testicular germ cell tumor tissue samples shows abundant tRNA fragments and global loss of piRNA. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-296. doi:10.1158/1538-7445.AM2015-LB-296

Abstract 232: Characterization of the human miRNA-ome reveals numerous novel, tissue-specific miRNAs that are implicated in cancer biology

Abstract Background: MicroRNAs (miRNAs) are short, ∼22 nt RNAs that are important post-transcriptional regulators of transcript abundance. Since their initial discovery they have been shown to be involved in many cellular processes and their dysregulation has been associated with disease etiology including many cancers. Currently, nearly 2,500 human miRNAs are annotated in public repositories. Key questions in miRNA research include: how many miRNAs are expressed in the human genome? What are their functional roles in various cancers? Herein, we sought to examine the first question through the examination of short RNA-sequence datasets. Methods: We analyzed 1,323 short RNA sequencing datasets (RNA-seq) generated from normal and disease samples representing 13 different human tissue types. We applied the miRDeep2 algorithm on each sample to identify short transcripts that are significantly expressed within the sample that are predicted to form hairpin-shaped secondary structures. Results: Using miRDeep2 with stringent thresholding criteria we discovered 3,707 statistically significant novel mature miRNAs at a false discovery rate of ≤ 0.05 that arise from 3,494 precursor loci: 91.5% of these novel miRNAs were discovered independently in 10 or more of the RNA-seq datasets. Analysis of the newly discovered miRNAs reveals that they are tissue-specific in nature. Furthermore, analysis of HITS-CLIP data independently obtained from four of the 13 tissues reveals that 45% of the novel miRNAs are loaded on Argonaute an indication that these miRNAs are active in the RNA interference pathway. Sequence conservation analysis across vertebrate and invertebrate organisms revealed that 56.7% of these new miRNAs are human-specific and the majority of them (94.4%) are primate-lineage specific. Importantly, our profiling of the newly discovered miRNAs showed their differential abundance across different tumor types, and between normal and tumor, and allowed tumor classification. Using orthogonal experimental techniques we have also been able to independently validate several dozen of the novel miRNAs. Conclusions: By analyzing samples from 13 human tissues we discovered 3,707 new miRNAs. This suggests that the repertoire of human miRNAs is larger and more diverse than might have been surmised by the publicly available data. We emphasize that the novel miRNAs we discovered are primate-specific and tissue-specific. This indicates the existence of molecular interactions that cannot be recapitulated by mouse models. As these interactions are numerous and currently uncharacterized they have consequential implications for both the clinical and basic research of cancers. Additionally, the fact that the abundance of our novel miRNAs can distinguish among different tumor types has direct implications for cancer biology and suggests possible uses of these novel molecules for therapeutic and diagnostic purposes. Citation Format: Eric R. Londin, Phillipe D. Loher, Isidore Rigoutsos. Characterization of the human miRNA-ome reveals numerous novel, tissue-specific miRNAs that are implicated in cancer biology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 232. doi:10.1158/1538-7445.AM2015-232

Two decades of miRNA biology: lessons and challenges.

The celebration of 20 years of RNA is opportune to reflect on what has been learned during this time regarding miRNA biology, and challenges that lay ahead. I focus this perspective on genetics, which arguably laid the foundation for the miRNA field. Indeed, genetic studies revealed critical miRNA:target interactions, the fundamental logic of miRNA target recognition, and phenotypically vital usages of miRNA regulation, all prior to formal recognition of the miRNA pathway. Ironically then, deciphering miRNA biology remains as challenging as ever, despite extensive mechanistic insights gained from biochemical and structural approaches, and a wealth of data garnered from genomewide approaches. Nevertheless, accumulating knowledge on miRNA function and ever-expanding genetic resources for manipulating miRNAs and their targets promise an exciting future in understanding in vivo necessities of these tiny RNAs. The miRNA era exploded in 2001 with the revelation that myriad hairpin-encoding loci generate ∼22 nucleotide (nt) RNAs. However, miRNA studies were already well on their way at that point, through developmental analyses in invertebrates. As is evident from their low-numbered gene names, mutants of the founding miRNAs lin-4 and let-7 were amongst the earliest C. elegans mutants recovered. Notably, the stage for comprehending their cloned sequences was set by genetic inquiries, initially by Chalfie and Ambros, which revealed antagonistic control of heterochronic identity by lin-4 and its major target lin-14. Ruvkun and colleagues studied gain-of-function lin-14 3′ UTR deletion mutants that phenocopied lin-4 loss-of-function, and these were critical to establish the cis-regulatory domain of lin-4 and its impact on lin-14 activity. However, there are older precedents for which loss of miRNA-mediated target regulation has phenotypic consequences. In 1923, Bridges isolated the dominant Drosophila wing serration mutant Beadex. Its eventual cloning showed that recurrent 3′ UTR disruptions underlie gain-of-function of dLMO. Interestingly, mir-9a knockouts exhibit similar wing serration, and miR-9a directly represses dLMO. Strikingly, removal of one dLMO allele fully restores mir-9a mutant wings, defining it as a critical miR-9a target. Thus, genetics revealed a miRNA-related phenotype nearly a century ago.

MicroRNAs: heralds of the noncoding RNA revolution.

MicroRNAs: heralds of the noncoding RNA revolution.

Identification of a gamma interferon-activated inhibitor of translation-like RNA motif at the 3' end of the transmissible gastroenteritis coronavirus genome modulating innate immune response.

ABSTRACTA 32-nucleotide (nt) RNA motif located at the 3′ end of the transmissible gastroenteritis coronavirus (TGEV) genome was found to specifically interact with the host proteins glutamyl-prolyl-tRNA synthetase (EPRS) and arginyl-tRNA synthetase (RRS). This RNA motif has high homology in sequence and secondary structure with the gamma interferon-activated inhibitor of translation (GAIT) element, which is located at the 3′ end of several mRNAs encoding proinflammatory proteins. The GAIT element is involved in the translation silencing of these mRNAs through its interaction with the GAIT complex (EPRS, heterogeneous nuclear ribonucleoprotein Q, ribosomal protein L13a, and glyceraldehyde 3-phosphate dehydrogenase) to favor the resolution of inflammation. Interestingly, we showed that the viral RNA motif bound the GAIT complex and inhibited the in vitro translation of a chimeric mRNA containing this RNA motif. To our knowledge, this is the first GAIT-like motif described in a positive RNA virus. To test the functional role of the GAIT-like RNA motif during TGEV infection, a recombinant coronavirus harboring mutations in this motif was engineered and characterized. Mutations of the GAIT-like RNA motif did not affect virus growth in cell cultures. However, an exacerbated innate immune response, mediated by the melanoma differentiation-associated gene 5 (MDA5) pathway, was observed in cells infected with the mutant virus compared with the response observed in cells infected with the parental virus. Furthermore, the mutant virus was more sensitive to beta interferon than the parental virus. All together, these data strongly suggested that the viral GAIT-like RNA motif modulates the host innate immune response.

A Solution to the C-Value Paradox and the Function of Junk DNA: The Genome Balance Hypothesis

The Genome Balance Hypothesis originated from a recent study that provided a mechanism for the phenomenon of genome dominance in ancient polyploids: unique 24nt RNA coverage near genes is greater in genes on the recessive subgenome irrespective of differences in gene expression. 24nt RNAs target transposons. Transposon position effects are now hypothesized to balance the expression of networked genes and provide spring-like tension between pericentromeric heterochromatin and microtubules. The balance (coordination) of gene expression and centromere movement is under selection. Our hypothesis states that this balance can be maintained by many or few transposons about equally well. We explain known balanced distributions of junk DNA within genomes and between subgenomes in allopolyploids (and our hypothesis passes “the onion test” for any so-called solution to the C-value paradox). Importantly, when the allotetraploid maize chromosomes delete redundant genes, their nearby transposons are also lost; this result is explained if transposons near genes function. The Genome Balance Hypothesis is hypothetical because the position effect mechanisms implicated are not proved to apply to all junk DNA, and the continuous nature of the centromeric and gene position effects have not yet been studied as a single phenomenon.

The genomes of many yam species contain transcriptionally active endogenous geminiviral sequences that may be functionally expressed.

Endogenous viral sequences are essentially ‘fossil records’ that can sometimes reveal the genomic features of long extinct virus species. Although numerous known instances exist of single-stranded DNA (ssDNA) genomes becoming stably integrated within the genomes of bacteria and animals, there remain very few examples of such integration events in plants. The best studied of these events are those which yielded the geminivirus-related DNA elements found within the nuclear genomes of various Nicotiana species. Although other ssDNA virus-like sequences are included within the draft genomes of various plant species, it is not entirely certain that these are not contaminants. The Nicotiana geminivirus-related DNA elements therefore remain the only definitively proven instances of endogenous plant ssDNA virus sequences. Here, we characterize two new classes of endogenous plant virus sequence that are also apparently derived from ancient geminiviruses in the genus Begomovirus. These two endogenous geminivirus-like elements (EGV1 and EGV2) are present in the Dioscorea spp. of the Enantiophyllum clade. We used fluorescence in situ hybridization to confirm that the EGV1 sequences are integrated in the D. alata genome and showed that one or two ancestral EGV sequences likely became integrated more than 1.4 million years ago during or before the diversification of the Asian and African Enantiophyllum Dioscorea spp. Unexpectedly, we found evidence of natural selection actively favouring the maintenance of EGV-expressed replication-associated protein (Rep) amino acid sequences, which clearly indicates that functional EGV Rep proteins were probably expressed for prolonged periods following endogenization. Further, the detection in D. alata of EGV gene transcripts, small 21–24 nt RNAs that are apparently derived from these transcripts, and expressed Rep proteins, provides evidence that some EGV genes are possibly still functionally expressed in at least some of the Enantiophyllum clade species.

Cross talk between ABC transporter mRNAs via a target mRNA-derived sponge of the GcvB small RNA.

There is an expanding list of examples by which one mRNA can posttranscriptionally influence the expression of others. This can involve RNA sponges that sequester regulatory RNAs of mRNAs in the same regulon, but the underlying molecular mechanism of such mRNA cross talk remains little understood. Here, we report sponge-mediated mRNA cross talk in the posttranscriptional network of GcvB, a conserved Hfq-dependent small RNA with one of the largest regulons known in bacteria. We show that mRNA decay from the gltIJKL locus encoding an amino acid ABC transporter generates a stable fragment (SroC) that base-pairs with GcvB. This interaction triggers the degradation of GcvB by RNase E, alleviating the GcvB-mediated mRNA repression of other amino acid-related transport and metabolic genes. Intriguingly, since the gltIJKL mRNA itself is a target of GcvB, the SroC sponge seems to enable both an internal feed-forward loop to activate its parental mRNA in cis and activation of many trans-encoded mRNAs in the same pathway. Disabling this mRNA cross talk affects bacterial growth when peptides are the sole carbon and nitrogen sources.

Identification of Regulatory-RNAs for Alternative Splicing of Ron Proto-Oncogene.

RON receptor tyrosine kinase is a proto-oncogene that induces cell migration and matrix invasion. RONΔ160 protein, which is produced by exclusion of exon 5 and 6, promotes cell migration, matrix invasion and protection from apoptosis. Alternative splicing regulation of exon 5 and 6 is not well understood. In this manuscript, we identified several new RNA regulatory elements for alternative splicing of Ron proto-oncogene. Firstly, we demonstrated that RNA sequences from EcoRI cleavage sites regulate alternative splicing of Ron exon 5 and 6. Secondly, we showed that the ~30 nt RNA at upstream end of exon 4 and the ~33 nt RNA at downstream end of exon 7 also modulate splicing of exon 5 and 6. Thirdly, our results indicate that the RNA sequences of the ends in exon 4 and 7 are required for the regulatory functions of the RNA from restriction enzyme cleavage sites. Our results provide a new insight for regulation of alternative splicing of Ron proto-oncogene.

Functional Analysis of the 5′-Regulatory Region of the Maize ALKALINE ALPHA-GALACTOSIDASE1 Gene

Maize ALKALINE ALPHA GALACTOSIDASE (ZmAGA) is a key enzyme in the raffinose metabolism pathway. We have previously characterized the ZmAGA1 gene as heat shock induced in germinating maize seeds. Here, we report that ZmAGA1 is induced in maize leaves by heat shock, dehydration, NaCl, abscisic acid (ABA), and salicylic acid. The 1622 bp of the 5′-regulatory region of the ZmAGA1 gene was isolated and characterized for promoter activity. The 5′-deletion assay of the regulatory region indicated that the region from −629 to −1 (relative to the start codon AUG) showed the greatest promoter activity, and the sequences upstream of this fragment act to inhibit transcription. The cis-acting element ABRE (ABA - responsive element) was identified to be functional. Increasingly, more refined 3′-deletion constructs of the regulatory region demonstrated that a 6-nucleotide (nt) RNA motif in the 5′-untranslated region (UTR) of the ZmAGA1 gene stimulates the translation of the downstream open reading frame (ORF). Deletion or triplication of this motif decreased or enhanced downstream ORF translation, respectively, while the mRNA abundance remained the same. RNA-EMSA (electrophoretic mobility shift assay) confirmed that some cytosolic protein(s) was (were) able to bind to this 6-nt RNA motif. This work contributes to our understanding of the ZmAGA1 gene regulatory mechanism and the metabolism of raffinose in maize cells.

Prediction and functional analysis of lincRNAs targeted by miRNAs.

Long intergenic non-coding RNAs (lincRNAs) located between protein-coding genes are non-coding RNAs longer than 200 nucleotides in length. LincRNAs are involved in a variety of biological processes such as cell cycle regulation, immune surveillance and embryonic stem cells differentiation in animals; however, the function of lincRNAs in plants is largely unclear. MicroRNAs (miRNAs) are a class of endogenous, single-stranded, non-coding small (~21 nt) RNAs, which can regulate gene expression at transcriptional or post-transcriptional level in eukaryotes by means of sequence complementation. Now, intensive studies on protein-coding genes targeted by miRNAs have been carried out, but the research on non-coding RNAs targeted by miRNAs is seldom explored, especially in plants. In order to uncover the potential function of lincRNAs, the data including miRNAs, cDNAs and degradomes were firstly collected and integrated, followed by bioinformatics methods to predict the potential binding sites of 337 mature miRNAs at 2708 lincRNAs in Arabidopsis thaliana. The regulatory networks of miRNAs-mRNAs-lincRNAs were constructed and the function of lincRNAs was predicted according to the competing endogenous RNA (ceRNA) hypothesis. This study may lay a solid foundation for elucidating the regulatory mechanism of miRNAs on lincRNAs as well as the function of lincRNAs in plants.

Breaking the protein-RNA recognition code

Breaking the protein-RNA recognition code

The role of miR-200a in mammalian epithelial cell transformation.

Cancer is a multistep disease that begins with malignant cell transformation and frequently culminates in metastasis. MicroRNAs (miRNAs) are small regulatory 21-25 nt RNA molecules and are frequently deregulated in cancer. miR-200a is a member of the miR-200 family, which are known inhibitors of the epithelial-to-mesenchymal transition. As such, the tumor-suppressive role of miR-200a in oncogenesis has been well documented; however, recent studies have found a proliferative role for this miRNA as well as a prometastatic role in the later steps of cancer progression. Little is known about the role of this miRNA in the early stages of cancer, namely, malignant cell transformation. Here, we show that miR-200a alone transforms an immortalized rat epithelial cell line, and miR-200a cooperates with Ras to enhance malignant transformation of an immortalized human epithelial cell line. Furthermore, miR-200a induces cell transformation and tumorigenesis in immunocompromised mice by cooperating with a Ras mutant that activates only the RalGEF effector pathway, but not Ras mutants activating PI3K or Raf effector pathways. This transformative ability is in accordance with miR-200a targeting Fog2 and p53 to activate Akt and directly repress p53 protein levels, respectively. These results demonstrate an oncogenic role for miR-200a and provide a specific cellular context where miR-200a acts as an oncomiR rather than a tumor suppressor by cooperating with an oncogene in malignant cell transformation.

RNA-Seq of the nucleolus reveals abundant SNORD44-derived small RNAs.

Small non-coding RNAs represent RNA species that are not translated to proteins, but which have diverse and broad functional activities in physiological and pathophysiological states. The knowledge of these small RNAs is rapidly expanding in part through the use of massive parallel (deep) sequencing efforts. We present here the first deep sequencing of small RNomes in subcellular compartments with particular emphasis on small RNAs (sRNA) associated with the nucleolus. The vast majority of the cellular, cytoplasmic and nuclear sRNAs were identified as miRNAs. In contrast, the nucleolar sRNAs had a unique size distribution consisting of 19–20 and 25 nt RNAs, which were predominantly composed of small snoRNA-derived box C/D RNAs (termed as sdRNA). Sequences from 47 sdRNAs were identified, which mapped to both 5′ and 3′ ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads mapping to SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as comprising both 20 and 25 nt RNAs. A novel 120 nt SNORD44 form was also identified. The expression of the SNORD44 sdRNA and 120 nt form was independent of Dicer/Drosha–mediated processing pathways but was dependent on the box C/D snoRNP proteins/sno-ribonucleoproteins fibrillarin and NOP58. The 120 nt SNORD44-derived RNA bound to fibrillarin suggesting that C/D sno-ribonucleoproteins are involved in regulating the stability or processing of SNORD44. This study reveals sRNA cell-compartment specific expression and the distinctive unique composition of the nucleolar sRNAs.

Multiplicity of 5' cap structures present on short RNAs.

Most RNA molecules are co- or post-transcriptionally modified to alter their chemical and functional properties to assist in their ultimate biological function. Among these modifications, the addition of 5′ cap structure has been found to regulate turnover and localization. Here we report a study of the cap structure of human short (<200 nt) RNAs (sRNAs), using sequencing of cDNA libraries prepared by enzymatic pretreatment of the sRNAs with cap sensitive-specificity, thin layer chromatographic (TLC) analyses of isolated cap structures and mass spectrometric analyses for validation of TLC analyses. Processed versions of snoRNAs and tRNAs sequences of less than 50 nt were observed in capped sRNA libraries, indicating additional processing and recapping of these annotated sRNAs biotypes. We report for the first time 2,7 dimethylguanosine in human sRNAs cap structures and surprisingly we find multiple type 0 cap structures (mGpppC, 7mGpppG, GpppG, GpppA, and 7mGpppA) in RNA length fractions shorter than 50 nt. Finally, we find the presence of additional uncharacterized cap structures that wait determination by the creation of needed reference compounds to be used in TLC analyses. These studies suggest the existence of novel biochemical pathways leading to the processing of primary and sRNAs and the modifications of their RNA 5′ ends with a spectrum of chemical modifications.

NMR resonance assignments of the archaeal ribosomal protein L7Ae in the apo form and bound to a 25 nt RNA.

The archaeal protein L7Ae forms part of a protein complex in the ribosome that specifically recognizes and binds to kink-turn RNA. In this complex, L7Ae directly interacts with the oligonucleotide and creates a functional arrangement for site-specific 2′-O-methylation. We report the solution NMR backbone assignment of Methanocaldococcus jannaschii L7Ae (117 residues, 12.7 kDa) in the ligand-free state and when bound to a 25 nucleotide C/D box kink-turn mimic RNA.