Omega speckles - a novel class of nuclear speckles containing hnRNPs associated with noncoding hsr-omega RNA in Drosophila

Fluorescence RNA:RNA in situ hybridization studies in various larval anti a dult cell types of Drosophila melanogaster showed that the noncoding hsr-om ega nuclear (hsr omega -n) transcripts were present in the form of many sma ll speckles. These speckles, which we name 'omega speckles', were distribut ed in the interchromatin space in close proximity to the chromatin. The onl y chromosomal site where hsr omega -n transcripts localized was the 93D loc us or the hsr omega gene itself, The number of nucleoplasmic speckles varie d in different cell types. Heat shock, which inhibits general chromosomal t ranscription, caused the individual speckles to coalesce into larger but fe wer clusters. In extreme cases, only a single large cluster of hsr omega -n transcripts localizing to the hsr omega locus was seen in each nucleus. In situ immunocytochemical staining using antibodies against heterogenous nuc lear RNA binding proteins (hnRNPs) like HRB87F, Hrp40, Hrb57A and SS reveal ed that, in all cell types, all the hnRNPs gave a diffuse staining of chrom atin areas and in addition, were present as large numbers of speckles. Colo calization studies revealed an absolute colocalization of the hnRNPs and th e omega speckles. Heat shock caused all the hnRNPs to cluster together exac tly, following the hsr omega -n transcripts. Immunoprecipitation studies us ing the hnRNP antibodies further demonstrated a physical association of hnR NPs and hsr omega transcripts. The omega speckles are distinct from interch romatin granules since nuclear speckles containing serine/arginine-rich SR- proteins like SC35 and SRp55 did not colocalize with the omega speckles. Th e speckled distribution of hnRNPs was completely disrupted in hsrw nullosom ics. We conclude that the hsr omega -n transcripts play essential structura l and functional roles in organizing and establishing the hnRNP-containing omega speckles and thus regulate the trafficking and availability of hnRNPs and other related RNA binding proteins in the cell nucleus.