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.
Highlights
The nucleolus contains a rich presentation of RNAs
The modification and folding of rRNAs is supported by numerous small nucleolar RNAs that are essential in guiding the proper positioning of rRNAs in large ribonucleoprotein (RNP) complexes [1,2,3]
The analysis indicated that most snoRNA-derived RNAs (sdRNA) reads showed a similar frequency of reads mapping to the 59 end of the small nucleolar RNAs (snoRNA) in both the nucleolar and nuclear fraction (e.g. SNORD44, SNORD105, SNORD57) (Fig. 3E)
Summary
The nucleolus contains a rich presentation of RNAs. Ribosomal (r) RNA biosynthesis comprises the main metabolic activity of the nucleolus. rRNA transcription is driven by a highly active dedicated polymerase, RNA polymerase I (Pol I), that transcribes rDNA genes to 47S precursor rRNA. The nucleolus contains a rich presentation of RNAs. Ribosomal (r) RNA biosynthesis comprises the main metabolic activity of the nucleolus. The 47S precursor is processed to the mature 28S, 18S and 5.8S RNAs by multiple steps that require the activity of proteins and enzymes for proper cleavage, modification and folding of the rRNAs. The modification and folding of rRNAs is supported by numerous small nucleolar RNAs (snoRNA) that are essential in guiding the proper positioning of rRNAs in large ribonucleoprotein (RNP) complexes [1,2,3]. The mature rRNAs are assembled to ribosomal 60S and 40S particles and translocated to the nucleus for further maturation [4]. This key metabolic activity, ribosome biogenesis, coordinates the assembly of the nucleolus into distinct subnucleolar domains that build around individual transcription and processing sites
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