RNA-Seq of the Nucleolus Reveals Abundant SNORD44-Derived Small RNAs

Abstract

Small non-coding RNAs represent RNA species that are not translated to proteins, but which have diverse and broad functional activities in physiological and pathophysiological states. The knowledge of these small RNAs is rapidly expanding in part through the use of massive parallel (deep) sequencing efforts. We present here the first deep sequencing of small RNomes in subcellular compartments with particular emphasis on small RNAs (sRNA) associated with the nucleolus. The vast majority of the cellular, cytoplasmic and nuclear sRNAs were identified as miRNAs. In contrast, the nucleolar sRNAs 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 to both 5′ and 3′ ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads mapping to SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as comprising both 20 and 25 nt RNAs. A novel 120 nt SNORD44 form was also identified. The expression of the SNORD44 sdRNA and 120 nt form was independent of Dicer/Drosha–mediated processing pathways but was dependent on the box C/D snoRNP proteins/sno-ribonucleoproteins fibrillarin and NOP58. The 120 nt SNORD44-derived RNA bound to fibrillarin suggesting that C/D sno-ribonucleoproteins are involved in regulating the stability or processing of SNORD44. This study reveals sRNA cell-compartment specific expression and the distinctive unique composition of the nucleolar sRNAs.

Dicer inactivation stimulates limb regeneration ability in Xenopus laevis

“…For normalization, same cDNAs were used as template to detect the expression of Ef1a, ornithine decarboxylase (ODC), or SNORD44, that can be used to quantitate ubiquitously expressed small nucleolar RNA as it's expression is independent of Dicer mediated pathways. 26 Normalization to Ef1a, odc, or SNORD44 produced similar result. Data shown in Figure 2H is from three biological replicates, normalized to Ef1a.…”

“…cDNAs were prepared with qScript microRNA cDNA synthesis kit (Quantabio, Beverly, MA), expression of miR‐196a and miR‐23a was analyzed with PerfeCTa Sybr green supermix (Quantabio), using microRNA assay primers together with PerfeCTa universal PCR primer provided by the supplier. For normalization, same cDNAs were used as template to detect the expression of Ef1a , ornithine decarboxylase ( ODC ), or SNORD44 , that can be used to quantitate ubiquitously expressed small nucleolar RNA as it's expression is independent of Dicer mediated pathways . Normalization to Ef1a , odc , or SNORD44 produced similar result.…”

Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system

“…This situation potentially changes their mode of activity and thus yielding different behaviors when functionally tested with the UTA reporter assays. In order to test this scenario, we used deep sequenced small RNAs from different subcellular compartments of HeLa cells from the GEO data base2845. To assure that the data retrieved and the conclusions we can generate are valid, we sequenced total small RNAs from HeLa cells and performed in parallel the same bioinformatics analysis to generate and compare retrieved fastq files.…”

“…HeLa cells small RNA data was retrieved from GEO GSE5005728, fastq files retrieved and generated were filtered using the Torrent Suite Software (Life Technologies, Carlsbad, USA) and high quality reads were then aligned to the miRbase21 and snoRNA base v3 using bowtie229, all the libraries were normalized using RPM (Reads assigned per million mapped reads) and used for plotting.…”

“…Fractionated cell compartment derived small RNA libraries were used for further analysis of nuclear, cytoplasmic and nucleolar content. ( A ) Experimental layout for HeLa small RNA library preparation in GSE5005728 and this study. ( B ) Comparison of small RNA expression profiles from HeLa cells from two independent sources, plot of normalized counts HeLa library prepared vs retrieved (r = 0.695, P < 2.2e16 r 2  = 0.48).…”