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Small RNA deep sequencing of cellular subcompartments

ABSTRACT: RNA deep sequencing efforts have revealed abundant expression of small RNAs derived from small nucleolar (sno) RNAs. We employ here spatial RNA deep sequencing to assess the expression of 10-40 nt small RNAs in subcellular compartments of HeLa cells. Total cellular, cytoplasmic, nuclear and nucleolar fractions were isolated, RNA was purified and size-fractionated in a two-step process to yield 10-40 nt RNA fractions. cDNA libraries were constructed and sequenced on a Ion Torrent platform. Vast majority of cellular, cytoplasmic and nuclear small RNA reads were identified as miRNAs. We found the expression of eleven ten miRNAs in the nucleolar preparations using a cut-off rate of 10 reads. Several miRNAs had a greater relative abundance in the nucleolus compared to the other compartments. The nucleolar small RNAs 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 both 5’ and 3’ ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads from SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as 20 and 25 nt RNAs. This study reveals a rich representation of cell-compartment specific expression of small RNAs and the distinctive unique composition of the nucleolar small RNAs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE50057 | GEO | 2014/05/07

SECONDARY ACCESSION(S): PRJNA215894

REPOSITORIES: GEO

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Small RNA deep sequencing of cellular subcompartments

Project description:RNA deep sequencing efforts have revealed abundant expression of small RNAs derived from small nucleolar (sno) RNAs. We employ here spatial RNA deep sequencing to assess the expression of 10-40 nt small RNAs in subcellular compartments of HeLa cells. Total cellular, cytoplasmic, nuclear and nucleolar fractions were isolated, RNA was purified and size-fractionated in a two-step process to yield 10-40 nt RNA fractions. cDNA libraries were constructed and sequenced on a Ion Torrent platform. Vast majority of cellular, cytoplasmic and nuclear small RNA reads were identified as miRNAs. We found the expression of eleven ten miRNAs in the nucleolar preparations using a cut-off rate of 10 reads. Several miRNAs had a greater relative abundance in the nucleolus compared to the other compartments. The nucleolar small RNAs 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 both 5M-bM-^@M-^Y and 3M-bM-^@M-^Y ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads from SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as 20 and 25 nt RNAs. This study reveals a rich representation of cell-compartment specific expression of small RNAs and the distinctive unique composition of the nucleolar small RNAs. Examination of small RNAs in cellular subcompartments

2014-05-07 | E-GEOD-50057 | biostudies-arrayexpress

small RNA shorter than 15 nt in commercial dairy cow's milk

Project description:We enlarged the usual 15-30 nt sequencing frame to 8-30 nt and discovered the existence in milk of 12-13 nt rRNA-derived small extracellular RNAs (exRNAs) previously reported in other datasets as doRNAs. We repeated the process on fractions of milk obtained by ultracentrifugation and found a specific enrichment of these unusually short rRNA between different milk fractions.

2022-04-02 | GSE199824 | GEO

Transcriptome Analysis of Differentially Expressed Genes in the Wild-type and Fabp7 KO Mouse Inner Ear

Project description:To reveal the function of FABP7 in the inner ear, we evaluated gene expressions between Wild-type and Fabp7 KO mice one day after noise exposure at 10 weeks, using RNA sequencing analysis. The read number was approximately 27–32 million paired-end reads per sample. A total of 24 and 23 genes were identified as upregulated and downregulated differentially expressed genes (DEGs), respectively in Fabp7 KO mice. The upregulation of genes related to nicotinamide-adenine dinucleotide (NADH) dehydrogenase (mt-Nd1, 2, 3, 4, and 6) and a glial high-affinity glutamate transporter (solute carrier family 1, member 2: Slc1a2) and downregulation of genes related to C/D box small nucleolar RNAs were observed in Fabp7 KO mice.

2023-11-22 | GSE241529 | GEO

Circulating miRNAs. isomiRs and small RNA clusters in human plasma and breast milk

Project description:Circulating small RNAs. including miRNAs but also isomiRs and other RNA species. have the potential to be used as non-invasive biomarkers for communicable and non-communicable diseases. This study aims to characterize miRNAs. isomiRs and small RNA clusters in biofluids. to compare their profiles. and to help in the establishment of standard guidelines. For this purpose. RNA from plasma and breast milk samples from 15 healthy postpartum mothers was extracted. Small RNA libraries were prepared with the NEBNext® small RNA library preparation kit and sequenced in an Illumina HiSeq2000 platform. After an initial quality control. miRNAs. isomiRs and clusters of small RNAs were annotated using seqBuster/seqCluster framework. The average amount of extracted RNA was 81 ng/mL [standard deviation (SD): 41] and 3985 ng/mL (SD: 3767) for plasma and breast milk. respectively. Mean number of good quality reads was 4.04 million (M) (40.01% of the reads) in plasma and 12.5M (89.6%) in breast milk. One thousand one hundred eighty two miRNAs. 74.317 isomiRs and 1.053 small RNA clusters that included piwi-interfering RNAs (piRNAs). tRNAs. small nucleolar RNAs (snoRNA) and small nuclear RNAs (snRNAs) were detected. Samples grouped by biofluid. with 308 miRNAs. 4.737 isomiRs and 778 small RNA clusters differentially detected. In summary. plasma and milk showed a completely different small RNA profile. In both. miRNAs. piRNAs. tRNAs. snRNAs. and snoRNAs were identified. confirming the presence of non-miRNA species in biofluids

2018-03-14 | GSE107524 | GEO

Identification and comprehensive analysis of microRNAs and phased small interfering RNAs in Rosa rugosa Thunb [ncRNA-seq]

Project description:MicroRNAs (miRNAs) are small non-coding RNAs that regulate target mRNAs by inducing degradation or preventing translation of their target mRNAs. Rose, Rosa rugosa Thunb., is an important ornamental and edible plant, yet there are only a few studies on the miRNAs of rose. Here we carried out computational and experimental analysis of miRNAs and phased small interfering RNAs (phasiRNAs) in rose by analyzing 10 small RNA profiles from roots, petals, pollens, stamens, and leaves. To identify the targets of miRNAs and phasiRNAs, we generated a degradome profile for rose leaf which is analyzed using the SeqTar algorithm. This study identified 25 conserved pre-miRNAs, of which 24 have not been reported previously. We also found 22 novel pre-miRNAs. Three hundred and thirty nine 21 nucleotide (nt) PHAS loci, and forty nine 24 nt PHAS loci were also identified. We identified more than 4000 putative targets of the conserved miRNAs using a criteria of less than 4 mismatches between miRNA and targets. Among these targets, at least 171 have shown significant accumulation of degradome reads. Our results demonstrate that the miR482 family triggers the generations of phasiRNAs by targeting nucleotide-binding, leucine-rich repeat (NB-LRR) disease resistance genes in rose. These results significantly enhanced our knowledge of the miRNAs and phasiRNAs, as well as their potential functions in rose.

2019-12-31 | GSE110490 | GEO

Identification and comprehensive analysis of microRNAs and phased small interfering RNAs in Rosa rugosa Thunb [degradome]

2019-12-31 | GSE110494 | GEO

ChIP-Seq of human endogenous small RNAs associated to Argonaute 1 and 2

Project description:Small non-coding RNAs function in concert with Argonaute (Ago) proteins to regulate gene expression at the level of transcription, mRNA stability or translation. Ago proteins bind small RNAs and form the core of silencing complexes. Here we report the analysis of small RNAs associated with human Ago1 and Ago2 revealed by immunoprecipitation and deep sequencing. Among the reads we find small RNAs originating from the small nucleolar RNA (snoRNA) ACA45. Moreover, processing of ACA45 requires Dicer activity but is independent of Drosha/DGCR8. Using bio-informatic prediction algorithms and luciferase reporter assays, we uncover the mediator subunit CDC2L6 as one potential mRNA target of ACA45 small RNAs suggesting a role for ACA45 processing products in post-transcriptional gene silencing. We further identify a number of human snoRNAs with microRNA (miRNA)- like processing signatures. We have therefore identified a novel class of small RNAs in human cells that originate from snoRNAs and can function like miRNAs. Two samples examined. Small RNAs associated to human Argonaute 1 and human Argonaute 2

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2022-11-01 | E-MTAB-12088 | biostudies-arrayexpress

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