Compartmentalization of RNA processing factors within nuclear speckles

In the mammalian cell nucleus pre-mRNA splicing factors are organized in a speckled pattern. The fluorescence signal within speckles appears homogeneo us when cells are immunolabeled with antibodies directed against pre-mRNA s plicing factors and examined by fluorescence microscopy. We have reexamined the speckled domains using serial dilutions of antibodies against SR prote ins, snRNPs, and a 3' end processing protein by immunofluorescence and conf ocal laser scanning microscopy. Using higher antibody dilutions, the speckl ed domains consist of numerous subdomains that are spherical. and heterogen eous in size ranging from 0.2 to 0.5 mu m in diameter. We refer to these su bdomains as "subspeckles." Each speckle is composed of 5 to 50 subspeckles and in some cases in actively transcribing cells, strings and loops of subs peckles were observed to extend from the speckled domains. Upon inhibition of RNA polymerase II transcription, the strings and loops of subspeckles we re no longer observed. Subspeckles were also not observed in coiled bodies. Using fluorescence in situ hybridization we found subspeckles to be coloca lized with transiently expressed beta-tropomyosin RNA transcripts. The comp artmentalization into subspeckles may represent an efficient way of organiz ing these factors for their subsequent transport to transcription/RNA proce ssing sites. (C) 2000 Academic Press.