Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:Purpose: The goal of this study is to compare the small RNA expression profile in exosomes isolated from plasma samples from KSHV+/HIV- and KSHV+/HIV+ patients Methods: Total RNA was isolated from plasma exosomes obtained from Ugandan patients with different viral status. A small RNA fraction was gel purified and was used to make sequencing libraries. At least 14 million read sequences were obtained from each library. Non-redondant sequences were then aligned to genomic (hg19) and and mRNA sequence (hg19) using bowtie 2; sequences with perfect-match and 1bp mismatch alignments were retained for further analysis using DEseq2 Methods: Total RNA was isolated from plasma obtained from KSHV-infected patients with or without HIV co-infection. A small RNA fraction was gel purified and was used to make sequencing libraries. At least 14 million read sequences were obtained from each library. Non-redondant sequences were then aligned to genomic (hg19) and and mRNA sequence (hg19) using bowtie 2; sequences with perfect-match and 1bp mismatch alignments were retained for further analysis using DEseq2 Results:Plasma exosomes isolated from either KSHV-infected or KSHV/ HIV co-infected patients exposed to malaria are overwhelmingly populated with YRNAs instead of miRNAs Results: KSHV oral sheeding correlated with the detection of a pro-tumorigenic cellualr miRNA profile in plasma. KSHV plasma viremia is associated with the detec tion of KSHV encoded miRNA in plasma Conclusions: Ugandan patients exposed to Plasmodium falciparum show an unusual repertoire of small RNAs in plasma exosomes, as the exosomal RNA is mostly composed of small RNA from the YRNA biotype Conclusions: Our study represents the first detailed analysis of the small RNA biotypes present in plasma from KSHV infected and KSHV/HIV co-infected patients without clinical sign of KSHV associated malignancies.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Transcriptional profiling of BJAB cells expressing miR-K10a and BCBL cells treated with miR-K10a inhibitor. To identify host RNA targets of KSHV miRNAs, we took advantage of the observation that RNAs targeted by miRNAs often display small reductions in their steady-state levels, perhaps as a result of their impaired translation. Accordingly, we examined cellular transcript accumulation by array-based expression profiling under three sets of conditions in which KSHV miRNAs were expressed or inhibited. Cells transfected with negative control miRNA compared to miR-K10a or negative miRNA inhibitor compared to miR-K10a inhibitor. The publication associated with this data set is about human TNFRSF12A/TWEAKR being targeted by KSHV miR-K10a.
Project description:Micro (mi)RNAs are small non-coding RNAs with key regulatory functions. Recent advances in the field allowed researchers to identify their targets. However, much less is known regarding the regulation of miRNA themselves. The accumulation of these tiny regulators can be modulated at various levels during their biogenesis from the transcription of the primary transcript (pri-miRNA) to the stability of the mature miRNA. Here, we studied the importance of the pri-miRNA secondary structure for the regulation of mature miRNAs accumulation. To this end, we used the KaposiM-bM-^@M-^Ys sarcoma herpesvirus, which encodes a cluster of twelve pre-miRNAs. Using small RNA profiling and quantitative northern blot analysis, we measured the absolute amount of each mature miRNAs in different cellular context. We found that the difference in expression between the least and most expressed viral miRNA could be as high as 60-fold. Using high-throughput selective 2M-bM-^@M-^Y-hydroxyl acylation analyzed by primer extension (hSHAPE), we then determined the secondary structure of the long primary transcript. We found that highly expressed miRNAs derived from optimally structured regions within the pri-miRNA. Finally, we confirmed the importance of the local structure by swapping stem-loops for highly and lowly expressed miRNAs, which resulted in a perturbed accumulation of the mature miRNA. Examination of sRNA profiles in 3 independent B cell lines expressing KSHV miRNAs or infected with KSHV, without replicate
Project description:Transcriptional profiling of BJAB cells expressing miR-K12-9 and BCBL cells treated with miR-K12-9 inhibitor. To identify host RNA targets of KSHV miRNAs, we took advantage of the observation that RNAs targeted by miRNAs often display small reductions in their steady-state levels, perhaps as a result of their impaired translation. Accordingly, we examined cellular transcript accumulation by array-based expression profiling under four sets of conditions in which KSHV miRNAs were expressed or inhibited. Cells transfected with negative control miRNA compared to miR-K12-9 or negative miRNA inhibitor compared to miR-K12-9 inhibitor.
Project description:Transcriptional profiling of BJAB cells expressing miR-K12-6-5p. To identify host RNA targets of KSHV miRNAs, we took advantage of the observation that RNAs targeted by miRNAs often display small reductions in their steady-state levels, perhaps as a result of their impaired translation. Accordingly, we examined cellular transcript accumulation by array-based expression profiling under four sets of conditions in which KSHV miRNAs were expressed. Cells transfected with negative control miRNA compared to miR-K12-6-5p.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes Sequence library of miRNAs from a single sample of human foetal mesenchymal stem cells. Results tested and confirmed by northern blotting. Please note that only raw data files are available for the embryonic and neual samples and thus, directly submitted to SRA (SRX547311, SRX548700, respectively under SRP042115/PRJNA247767)