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.