Characterization of a naturally occurring truncated Dicer.
Dicer is central to small RNA silencing pathways, thus playing an important role in physiological and pathological states. Recently, a number of mutations in dicer gene have been identified in diverse types of cancer, implicating Dicer in oncogenic cooperation. Here we report on the properties of a rare splice variant of the human dicer gene, occurring in neuroblastoma cells, and not detectable in normal tissues. Due to the skipping of one exon, the alternatively spliced transcript encodes a putative truncated protein, t-Dicer, lacking the dsRNA-binding domain and bearing altered one of the two RNase III catalytic centers. The ability of the exon-depleted t-dicer transcript to be translated in vitro was first investigated by the expression of flagged t-Dicer in human cells. We found that t-dicer transcript could be translated in vitro, albeit not as efficiently as full-length dicer transcript. Then, the possible enzymatic activity of t-Dicer was analyzed by an in vitro dicing assay able to distinguish the enzymatic activity of the individual RNase III domains. We showed that t-Dicer preserved partial dicing activity. Overall, the results indicate that t-dicer transcript could produce a protein still able to bind the substrate and to cleave only one of the two pre-miRNA strands. Given the increasing number of mutations reported for dicer gene in tumours, our experimental approach could be useful to characterize the activity of these mutants, which may dictate changes in selected classes of small RNAs and/or lead to their aberrant maturation.
Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, via Vivaldi 43, 81100, Caserta, Italy.
Nicola
N
Mosca
Julia
J
Starega-Roslan
Filomena
F
Castiello
Aniello
A
Russo
Wlodzimierz J
WJ
Krzyzosiak
Nicoletta
N
Potenza
D017398:Alternative Splicing; D020134:Catalytic Domain; D057075:Enzyme Assays; D005091:Exons; D015870:Gene Expression; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D009447:Neuroblastoma / Q000201:enzymology* / Q000235:genetics; D043244:Ribonuclease III / Q000235:genetics* / Q000378:metabolism
D035683:MicroRNAs; D043244:Ribonuclease III