Abstract
BackgroundShort non-coding RNAs (ncRNAs) perform their cellular functions in ribonucleoprotein (RNP) complexes, which are also essential for maintaining the stability of the ncRNAs. Depletion of individual protein components of non-coding ribonucleoprotein (ncRNP) particles by RNA interference (RNAi) may therefore affect expression levels of the corresponding ncRNA, and depletion of candidate associated proteins may constitute an alternative strategy when investigating ncRNA-protein interactions and ncRNA functions. Therefore, we carried out a pilot study in which the effects of RNAi against protein components of small nucleolar RNPs (snoRNPs) in Caenorhabditis elegans were observed on an ncRNA microarray.ResultsRNAi against individual C. elegans protein components of snoRNPs produced strongly reduced mRNA levels and distinct phenotypes for all targeted proteins. For each type of snoRNP, individual depletion of at least three of the four protein components produced significant (P ≦ 1.2 × 10-5) reductions in the expression levels of the corresponding small nucleolar RNAs (snoRNAs), whereas the expression levels of other ncRNAs were largely unaffected. The effects of depletion of individual proteins were in accordance with snoRNP structure analyses obtained in other species for all but two of the eight targeted proteins. Variations in snoRNA size, sequence and secondary structure characteristics were not systematically reflected in the affinity for individual protein component of snoRNPs. The data supported the classification of nearly all annotated snoRNAs and suggested the presence of several novel snoRNAs among unclassified short ncRNA transcripts. A number of transcripts containing canonical Sm binding element sequences (Sm Y RNAs) also showed reduced expression after depletion of protein components of C/D box snoRNPs, whereas the expression of some stem-bulge RNAs (sbRNAs) was increased after depletion of the same proteins.ConclusionThe study confirms observations made for other organisms, where reduced ncRNA levels after depletion of protein components of ncRNPs were noted, and shows that such reductions in expression levels occur across entire sets of ncRNA. Thereby, the study also demonstrates the feasibility of combining RNAi against candidate proteins with ncRNA microarray analysis to investigate ncRNA-protein interactions and hence ncRNA cellular functions.
Highlights
Short non-coding RNAs perform their cellular functions in ribonucleoprotein (RNP) complexes, which are essential for maintaining the stability of the ncRNAs
If proteins belonging to specific complexes or pathways are depleted by RNA interference (RNAi), which in C. elegans can be achieved by feeding worms with E. coli expressing double-stranded RNA corresponding to fragments of the targeted protein gene, effects on the ncRNA population can be observed with a designed microarray, and ncRNA-protein interactions subsequently inferred from the reduced expression levels of associated ncRNAs
In order to observe the effects on the ncRNA expression levels, we carried out RNAi against protein components of the corresponding non-coding ribonucleoprotein (ncRNP)
Summary
Short non-coding RNAs (ncRNAs) perform their cellular functions in ribonucleoprotein (RNP) complexes, which are essential for maintaining the stability of the ncRNAs. Tests in our lab (data not shown) suggest that C. elegans short ncRNAs are recalcitrant to knock-down by RNAi, irrespective of ncRNA class or mode of RNAi application, thereby rendering unavailable a potentially efficient tool for functional analysis of such transcripts It was observed, though, that RNAi depletion of individual protein components of ncRNA complexes in a number of cases would substantially reduce the expression levels of the associated ncRNAs. It was observed, though, that RNAi depletion of individual protein components of ncRNA complexes in a number of cases would substantially reduce the expression levels of the associated ncRNAs Such effects have been reported for small nucleolar RNAs (snoRNAs) in yeast [8,9,10,11,12], and a similar approach has been used to analyse the potential involvement of telomerase RNA in human cancers [13]. The study will contribute to correct annotation of transcripts with indistinct snoRNA characteristics, an issue that was raised in a recent study [15]
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