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A hyperphosphorylated form of the largest subunit of RNA polymerase II (pol IIo) is associated with the pre-mRNA splicing process. Pol IIo was detected in association with a subset of small nuclear ribonucleoprotein particle and Ser-Arg protein splicing factors and also with pre-mRNA splicing complexes assembled in vitro. A subpopulation of pol hIo was localized to nuclear "speckle" domains enriched in splicing factors, indicating that it may also be associated with RNA processing in vivo. Moreover, pol IIo was retained in a similar pattern following in situ extraction of cells and was quantitatively recovered in the nuclear matrix fraction. The results implicate nuclear matrix-associated hyperphosphorylated pol IIo as a possible link in the coordination of transcription and splicing processes.with pre-mRNA processing that are related to the SR family.In the present study, a new anti-NM mAb, B3, is characterized that recognizes a 250-kDa NM protein concentrated in speckles. Similar to anti-NM mAbs which recognize SR proteins, B3 preferentially binds in vitro to a subset of splicing complexes containing exon sequences. Surprisingly, the B3 antigen corresponds to a hyperphosphorylated form of the large subunit of pol II (pol IIo). In addition to splicing complexes, pol IIo is associated with a subset of snRNP and SR protein splicing factors. The possible implications of these findings in relation to the regulation of RNA processing are discussed. MATERIALS AND METHODSIncreasing evidence suggests that transcription and processing of RNA polymerase II (pol II) transcripts are temporally and spatially linked. Visualization of chromatin spreads by electron microscopy has revealed that the majority of introns are removed cotranscriptionally from pre-mRNA (1, 2). These studies are supported by recent fluorescent in situ hybridization experiments, indicating that the synthesis and splicing of specific pol II transcripts are coincident at discrete foci (3-5).In several cases, transcript foci appear to be localized in association with specific nuclear domains that are highly enriched in splicing factors, referred to as "speckles" (3, 5-7).Although not mutually exclusive with evidence implicating speckle domains in splicing factor storage and/or assembly (8, 9), these transcript localization experiments indicate a possible direct role of speckle domains in the processing of pre-mRNAs (10, 11). Mammalian nuclei typically contain 20-50 speckle domains, which, in addition to the four spliceosomal small nuclear ribonucleoprotein particles (snRNPs; Ul, U2, U4/6, and U5), are also enriched for non-snRNP splicing factors and poly(A)+ RNA (8, 9, 11). Many of the non-snRNP splicing factors in speckles are related to the Ser-Arg (SR) family of proteins, all of which contain one or more domains rich in alternating serine and arginine residues (12). Besides splicing components, speckle structures also contain elevated concentrations of proteins involved in transcription and cellular transformation (13-15). Since these structure...

Microscopy shows that individual sites of DNA replication and transcription of mammalian nuclei segregate into sets of roughly 22 and 16 higher order domains, respectively. Each domain set displayed a distinct network-like appearance, including regions of individual domains and interdigitation of domains between the two networks. These data support a dynamic mosaic model for the higher order arrangement of genomic function inside the cell nuclei.

A layered organization of cells is a common architectural feature of many neuronal formations. Mutations of the zebrafish gene nagie oko (nok) produce a severe disruption of retinal architecture, indicating a key role for this locus in neuronal patterning. We show that nok encodes a membrane-associated guanylate kinase-family scaffolding protein. Nok localizes to the vicinity of junctional complexes in retinal neuroepithelium and in the photoreceptor cell layer. Mosaic analysis indicates that the nok retinal patterning phenotype is not cell-autonomous. We propose that nok function in patterning of postmitotic neurons is mediated through neuroepithelial cells and is necessary for guiding neurons to their proper destinations in retinal laminae.