Efficient and quantitative high-throughput transfer RNA sequencing
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Tao Pan
PROVIDER: E-GEOD-66550 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The developmentally regulated 26- to 32-nt siRNAs (scnRNAs) are loaded to the Argonaute protein Twi1p and display a strong bias for uracil at the 5' end. In this study, we used deep sequencing to analyze loaded and unloaded populations of scnRNAs. We show that the size of the scnRNA is determined during a pre-loading process, whereas their 5' uracil bias is attributed to both pre-loading and loading processes. We also demonstrate that scnRNAs have a strong bias for adenine at the third base from the 3' terminus, suggesting that most scnRNAs are direct Dicer products. Furthermore, we show that the thermodynamic asymmetry of the scnRNA duplex does not affect the guide and passenger strand decision. Finally, we show that scnRNAs frequently have templated uracil at the last base without a strong bias for adenine at the second base indicating non-sequential production of scnRNAs from substrates. These findings provide a biochemical basis for the varying attributes of scnRNAs, which should help improve our understanding of the production and turnover of scnRNAs in vivo. We compared Twi1p-loaded scnRNAs to scnRNAs before they have been loaded into Twi1p by deep sequencing to understand how the two processes, the production of siRNAs by Dicer and the loading of siRNAs into Argonaute, shape the population of siRNAs in vivo.
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Project description:The assembly of fission yeast pericentromeric heterochromatin and generation of small interfering RNAs (siRNAs) from noncoding centromeric transcripts are mutually dependent processes. How this interdependent positive feedback loop is first triggered is a fundamental unanswered question. Here we show that two distinct Argonaute (Ago1)-dependent pathways mediate small RNA generation. RNA-dependent RNA polymerase complex (RDRC) and Dicer act on specific noncoding RNAs to generate siRNAs by a mechanism that requires the slicer activity of Ago1 but is independent of pre-existing heterochromatin. In the absence of RDRC or Dicer, a distinct class of small RNAs, called primal small RNAs (priRNAs), associate with Ago1. priRNAs are degradation products of abundant transcripts, which bind to Ago1 and target antisense transcripts that result from bidirectional transcription of DNA repeats. Our results suggest that a transcriptome surveillance mechanism based on the random association of RNA degradation products with Argonaute triggers siRNA amplification and heterochromatin assembly within DNA repeats. small RNA profiling in wild type S. pombe cells and in 12 mutant cells
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Project description:The transposon silencing piRNAs are produced from precursors that are encoded by heterochromatic clusters and processed in the perinuclear nuage. We show that the Drosophila nuclear DEAD box protein UAP56, previously implicated in mRNA splicing and nuclear export, co-localizes with the cluster-associated HP1 homologue Rhino. Prominent nuclear foci containing Rhi and UAP56 localize directly across the nuclear envelope from Vasa, a conserved DEAD box protein and core nuage component that is required for piRNA production, and piRNA precursors immunoprecipitate with both UAP56 and Vasa. A uap56 point mutation that prevents UAP56 protein co-localization with Rhino also disrupts nuage organization, transposon silencing, and expression of dual strand piRNA clusters. By contrast, this allele significantly increases ectopic piRNAs from protein coding genes. We therefore propose that UAP56 and Vasa organize a piRNA-processing compartment that spans the nuclear envelope, increasing the efficiency and specificity of piRNA biogenesis. RNA-Seq: 3 samples examined: w1118, uap56 mutant un-oxidized, uap56 mutant oxidized RIP-Seq: 6 samples: UAP56-Venus, sz-Venus, and wild type w1 with anti-flag and input control each.
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