RNA-based affinity purification reveals 7SK RNPs with distinct composition and regulation

Recent studies have uncovered an unanticipated diversity of noncoding RNAs (ncRNAs), although these studies provide limited insight into their biological significance. Numerous general methods for identification and characterization of protein interactions have been developed, but similar approaches for characterizing cellular ncRNA interactions are lacking. Here we describe RNA Affinity in Tandem (RAT), an original, entirely RNA tag-based method for affinity purification of endogenously assembled RNP complexes. We demonstrate the general utility of RAT by isolating RNPs assembled in vivo on ncRNAs transcribed by RNA polymerase II or III. Using RAT in conjunction with protein identification by mass spectrometry and protein-RNA interaction assays, we define and characterize previously unanticipated protein subunits of endogenously assembled human 7SK RNPs. We show that 7SK RNA resides in a mixed population of RNPs with different protein compositions and responses to cellular stress. Depletion of a newly identified 7SK RNP component, hnRNP K, alters the partitioning of 7SK RNA among distinct RNPs. Our results establish the utility of a generalizable RNA-based RNP affinity purification method and provide insight into 7SK RNP dynamics.     

Identification of small non-coding RNAs from mitochondria and chloroplasts

Small non-protein-coding RNAs (ncRNAs) have been identified in a wide spectrum of organisms ranging from bacteria to humans. In eukarya, systematic searches for ncRNAs have so far been restricted to the nuclear or cytosolic compartments of cells. Whether or not small stable non-coding RNA species also exist in cell organelles, in addition to tRNAs or ribosomal RNAs, is unknown. We have thus generated cDNA libraries from size-selected mammalian mitochondrial RNA and plant chloroplast RNA and searched for small ncRNA species in these two types of DNA-containing cell organelles. In total, we have identified 18 novel candidates for organellar ncRNAs in these two cellular compartments and confirmed expression of six of them by northern blot analysis or RNase A protection assays. Most candidate ncRNA genes map to intergenic regions of the organellar genomes. As found previously in bacteria, the presumptive ancestors of present-day chloroplasts and mitochondria, we also observed examples of antisense ncRNAs that potentially could target organelle-encoded mRNAs. The structural features of the identified ncRNAs as well as their possible cellular functions are discussed. The absence from our libraries of abundant small RNA species that are not encoded by the organellar genomes suggests that the import of RNAs into cell organelles is of very limited significance or does not occur at all.     

Generation of a neuro-specific microarray reveals novel differentially expressed noncoding RNAs in mouse models for neurodegenerative diseases

We have generated a novel, neuro-specific ncRNA microarray, covering 1472 ncRNA species, to investigate their expression in different mouse models for central nervous system diseases. Thereby, we analyzed ncRNA expression in two mouse models with impaired calcium channel activity, implicated in Epilepsy or Parkinson''s disease, respectively, as well as in a mouse model mimicking pathophysiological aspects of Alzheimer''s disease. We identified well over a hundred differentially expressed ncRNAs, either from known classes of ncRNAs, such as miRNAs or snoRNAs or which represented entirely novel ncRNA species. Several differentially expressed ncRNAs in the calcium channel mouse models were assigned as miRNAs and target genes involved in calcium signaling, thus suggesting feedback regulation of miRNAs by calcium signaling. In the Alzheimer mouse model, we identified two snoRNAs, whose expression was deregulated prior to amyloid plaque formation. Interestingly, the presence of snoRNAs could be detected in cerebral spine fluid samples in humans, thus potentially serving as early diagnostic markers for Alzheimer''s disease. In addition to known ncRNAs species, we also identified 63 differentially expressed, entirely novel ncRNA candidates, located in intronic or intergenic regions of the mouse genome, genomic locations, which previously have been shown to harbor the majority of functional ncRNAs.     

Genome-wide searching with base-pairing kernel functions for noncoding RNAs: computational and expression analysis of snoRNA families in Caenorhabditis elegans

Despite the accumulating research on noncoding RNAs (ncRNAs), it is likely that we are seeing only the tip of the iceberg regarding our understanding of the functions and the regulatory roles served by ncRNAs in cellular metabolism, pathogenesis and host-pathogen interactions. Therefore, more powerful computational and experimental tools for analyzing ncRNAs need to be developed. To this end, we propose novel kernel functions, called base-pairing profile local alignment (BPLA) kernels, for analyzing functional ncRNA sequences using support vector machines (SVMs). We extend the local alignment kernels for amino acid sequences in order to handle RNA sequences by using STRAL's; scoring function, which takes into account sequence similarities as well as upstream and downstream base-pairing probabilities, thus enabling us to model secondary structures of RNA sequences. As a test of the performance of BPLA kernels, we applied our kernels to the problem of discriminating members of an RNA family from nonmembers using SVMs. The results indicated that the discrimination ability of our kernels is stronger than that of other existing methods. Furthermore, we demonstrated the applicability of our kernels to the problem of genome-wide search of snoRNA families in the Caenorhabditis elegans genome, and confirmed that the expression is valid in 14 out of 48 of our predicted candidates by using qRT-PCR. Finally, highly expressed six candidates were identified as the original target regions by DNA sequencing.     

Interaction cloning and characterization of RoBPI, a novel protein binding to human Ro ribonucleoproteins

Human Ro ribonucleoproteins (RNPs) are autoantigenic particles of unknown function(s) that consist of a 60-kDa protein (Ro60) associated with one hY RNA (hY1-5). Using a modified yeast three-hybrid system, named RNP interaction trap assay (RITA), we cloned a novel Ro RNP-binding protein (RoBPI), based on its property to interact in vivo in yeast with an RNP complex made of recombinant Ro60 (rRo60) protein and hY5 (rhY5) RNA. RoBPI cDNA contains three conserved RNA recognition motifs (RRM) and is present as a family of isoforms differing slightly at their 5' end. The 2.0-kb RoBPI mRNA was detected in all human tissues tested. Highly homologous cDNA sequences were found in banks of expressed sequence tags (ESTs) from mice. Two-hybrid, three-hybrid, and RITA experiments respectively established that 60 kDa RoBPI did not interact in yeast with rRo60 alone, with rhY5 RNA alone, or with bait RNPs consisting of rRo60 and recombinant hY1, hY3, or hY4 RNAs. RoBPI coimmunoprecipitated with Ro RNPs from HeLa cell extracts and partially colocalized with Ro60 in nuclei of cultured cells. Because hY5 RNA and RohY5 RNPs are recent evolutionary additions seen only in primates, but RoBPI seems more conserved, their interaction may represent a gain of function for Ro RNPs. Alternatively, interaction of RohY5 RNPs with RoBPI may have no functional bearing, but may underlie some of the unique biochemical and immunological properties of these RNPs.     

smyRNA: A Novel Ab Initio ncRNA Gene Finder

Background

Non-coding RNAs (ncRNAs) have important functional roles in the cell: for example, they regulate gene expression by means of establishing stable joint structures with target mRNAs via complementary sequence motifs. Sequence motifs are also important determinants of the structure of ncRNAs. Although ncRNAs are abundant, discovering novel ncRNAs on genome sequences has proven to be a hard task; in particular past attempts for ab initio ncRNA search mostly failed with the exception of tools that can identify micro RNAs.

Methodology/Principal Findings

We present a very general ab initio ncRNA gene finder that exploits differential distributions of sequence motifs between ncRNAs and background genome sequences.

Conclusions/Significance

Our method, once trained on a set of ncRNAs from a given species, can be applied to a genome sequences of other organisms to find not only ncRNAs homologous to those in the training set but also others that potentially belong to novel (and perhaps unknown) ncRNA families. Availability: http://compbio.cs.sfu.ca/taverna/smyrna     

Sequence complementarity between human noncoding RNAs and SARS-CoV-2 genes: What are the implications for human health?

ObjectivesTo investigate in silico the presence of nucleotide sequence complementarity between the RNA genome of Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) and human non-coding (nc)RNA genes.MethodsThe FASTA sequence (NC_045512.2) of each of the 11 SARS-CoV-2 isolate Wuhan-Hu-1 genes was retrieved from NCBI.nlm.nih.gov/gene and the Ensembl.org library interrogated for any base-pair match with human ncRNA genes. SARS-CoV-2 gene-matched human ncRNAs were screened for functional activity using bioinformatic analysis. Finally, associations between identified ncRNAs and human diseases were searched in GWAS databases.ResultsA total of 252 matches were found between the nucleotide sequence of SARS-CoV-2 genes and human ncRNAs. With the exception of two small nuclear RNAs, all of them were long non-coding (lnc)RNAs expressed mainly in testis and central nervous system under physiological conditions. The percentage of alignment ranged from 91.30% to 100% with a mean nucleotide alignment length of 17.5 ± 2.4. Thirty-three (13.09%) of them contained predicted R-loop forming sequences, but none of these intersected the complementary sequences of SARS-CoV-2. However, in 31 cases matches fell on ncRNA regulatory sites, whose adjacent coding genes are mostly involved in cancer, immunological and neurological pathways. Similarly, several polymorphic variants of detected non-coding genes have been associated with neuropsychiatric and proliferative disorders.ConclusionThis pivotal in silico study shows that SARS-CoV-2 genes have Watson-Crick nucleotide complementarity to human ncRNA sequences, potentially disrupting ncRNA epigenetic control of target genes. It remains to be elucidated whether this could result in the development of human disease in the long term.     

Analyzing modular RNA structure reveals low global structural entropy in microRNA sequence

Secondary structure remains the most exploitable feature for noncoding RNA (ncRNA) gene finding in genomes. However, methods based on secondary structure prediction may generate superfluous amount of candidates for validation and have yet to deliver the desired performance that can complement experimental efforts in ncRNA gene finding. This paper investigates a novel method, unpaired structural entropy (USE) as a measurement for the structure fold stability of ncRNAs. USE proves to be effective in identifying from the genome background a class of ncRNAs, such as precursor microRNAs (pre-miRNAs) that contains a long stem hairpin loop. USE correlates well and performs better than other measures on pre-miRNAs, including the previously formulated structural entropy. As an SVM classifier, USE outperforms existing pre-miRNA classifiers. A long stem hairpin loop is common for a number of other functional RNAs including introns splicing hairpins loops and intrinsic termination hairpin loops. We believe USE can be further applied in developing ab initio prediction programs for a larger class of ncRNAs.     

Plant noncoding RNA gene discovery by "single-genome comparative genomics"

Plant genomes have undergone multiple rounds of duplications that contributed massively to the growth of gene families. The structure of resulting families has been studied in depth for protein-coding genes. However, little is known about the impact of duplications on noncoding RNA (ncRNA) genes. Here we perform a systematic analysis of duplicated regions in the rice genome in search of such ncRNA repeats. We observe that, just like their protein counterparts, most ncRNA genes have undergone multiple duplications that left visible sequence conservation footprints. The extent of ncRNA gene duplication in plants is such that these sequence footprints can be exploited for the discovery of novel ncRNA gene families on a large scale. We developed an SVM model that is able to retrieve likely ncRNA candidates among the 100,000+ repeat families in the rice genome, with a reasonably low false-positive discovery rate. Among the nearly 4000 ncRNA families predicted by this means, only 90 correspond to putative snoRNA or miRNA families. About half of the remaining families are classified as structured RNAs. New candidate ncRNAs are particularly enriched in UTR and intronic regions. Interestingly, 89% of the putative ncRNA families do not produce a detectable signal when their sequences are compared to another grass genome such as maize. Our results show that a large fraction of rice ncRNA genes are present in multiple copies and are species-specific or of recent origin. Intragenome comparison is a unique and potent source for the computational annotation of this major class of ncRNA.