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Functional profiling of long intergenic non-coding RNAs in fission yeast.

ABSTRACT: Eukaryotic genomes express numerous long intergenic non-coding RNAs (lincRNAs) that do not overlap any coding genes. Some lincRNAs function in various aspects of gene regulation, but it is not clear in general to what extent lincRNAs contribute to the information flow from genotype to phenotype. To explore this question, we systematically analysed cellular roles of lincRNAs in Schizosaccharomyces pombe. Using seamless CRISPR/Cas9-based genome editing, we deleted 141 lincRNA genes to broadly phenotype these mutants, together with 238 diverse coding-gene mutants for functional context. We applied high-throughput colony-based assays to determine mutant growth and viability in benign conditions and in response to 145 different nutrient, drug, and stress conditions. These analyses uncovered phenotypes for 47.5% of the lincRNAs and 96% of the protein-coding genes. For 110 lincRNA mutants, we also performed high-throughput microscopy and flow cytometry assays, linking 37% of these lincRNAs with cell-size and/or cell-cycle control. With all assays combined, we detected phenotypes for 84 (59.6%) of all lincRNA deletion mutants tested. For complementary functional inference, we analysed colony growth of strains ectopically overexpressing 113 lincRNA genes under 47 different conditions. Of these overexpression strains, 102 (90.3%) showed altered growth under certain conditions. Clustering analyses provided further functional clues and relationships for some of the lincRNAs. These rich phenomics datasets associate lincRNA mutants with hundreds of phenotypes, indicating that most of the lincRNAs analysed exert cellular functions in specific environmental or physiological contexts. This study provides groundwork to further dissect the roles of these lincRNAs in the relevant conditions.

SUBMITTER: Rodriguez-Lopez M

PROVIDER: S-EPMC8730722 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Long non-coding RNAs (lncRNAs) are key regulators of diverse cellular processes. Although a number of studies have reported the identification of bovine lncRNAs across many tissues, very little is known about the identity and characteristics of lncRNAs in bovine oocytes. METHODS:A bovine oocyte cDNA library was constructed and sequenced using the Illumina HiSeq 2000 sequencing system. The oocyte transcriptome was constructed using the ab initio assembly software Scripture and Cufflinks. The assembled transcripts were categorized to identify the novel intergenic transcripts, and the coding potential of these novel transcripts was assessed using CPAT and PhyloCSF. The resulting candidate long intergenic non-coding RNAs (lincRNAs) transcripts were further evaluated to determine if any of them contain any known protein coding domains in the Pfam database. RT-PCR was used to analyze the expression of oocyte-expressed lincRNAs in various bovine tissues. RESULTS:A total of 85 million raw reads were generated from sequencing of the bovine oocyte library. Transcriptome reconstruction resulted in the assembly of a total of 42,396 transcripts from 37,678 genomic loci. Analysis of the assembled transcripts using the step-wide pipeline resulted in the identification of 1535 oocyte lincRNAs corresponding to 1183 putative non-coding genes. A comparison of the oocyte lincRNAs with the lncRNAs reported in other bovine tissues indicated that 970 of the 1535 oocyte lincRNAs appear to be unique to bovine oocytes. RT-PCR analysis of 5 selected lincRNAs showed either specific or predominant expression of 4 lincRNAs in the fetal ovary. Functional prediction of the oocyte-expressed lincRNAs suggested their involvement in oogenesis through regulating their neighboring protein-coding genes. CONCLUSIONS:This study provides a starting point for future research aimed at understanding the roles of lncRNAs in controlling oocyte development and early embryogenesis in cattle.

Project description:Recently, large numbers of normal human tissues have been profiled for non-coding RNAs and more than fourteen thousand long intergenic non-coding RNAs (lincRNAs) are found expressed in normal human tissues. The functional roles of these normal lincRNAs (nlincRNAs) in the regulation of protein coding genes in normal and disease biology are yet to be established. Here, we have profiled two RNA-seq datasets including cancer and matched non-neoplastic tissues from 12 individuals from diverse demography for both coding genes and nlincRNAs. We find 130 nlincRNAs significantly regulated in cancer, with 127 regulated in the same direction in the two datasets. Interestingly, according to Illumina Body Map, significant numbers of these nlincRNAs display baseline null expression in normal prostate tissues but are specific to other tissues such as thyroid, kidney, liver and testis. A number of the regulated nlincRNAs share loci with coding genes, which are either co-regulated or oppositely regulated in all cancer samples studied here. For example, in all cancer samples i) the nlincRNA, TCONS_00029157, and a neighboring tumor suppressor factor, SIK1, are both down regulated; ii) several thyroid-specific nlincRNAs in the neighborhood of the thyroid-specific gene TPO, are both up-regulated; and iii) the TCONS_00010581, an isoform of HEIH, is down-regulated while the neighboring EZH2 gene is up-regulated in cancer. Several nlincRNAs from a prostate cancer associated chromosomal locus, 8q24, are up-regulated in cancer along with other known prostate cancer associated genes including PCAT-1, PVT1, and PCAT-92. We observe that there is significant bias towards up-regulation of nlincRNAs with as high as 118 out of 127 up-regulated in cancer, even though regulation of coding genes is skewed towards down-regulation. Considering that all reported cancer associated lincRNAs (clincRNAs) are biased towards up-regulation, we conclude that this bias may be functionally relevant.

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Functional profiling of long intergenic non-coding RNAs in fission yeast.

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