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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...

We report the identification and cloning of a nuclear matrix protein termed matrin cyclophilin or matrin CYP. The derived sequence of matrin cyp encodes a protein of 752 amino acids with a predicted mass of 88 kDa. A 172-residue stretch at the amino terminus shows high identity with the ubiquitous family of cyclophilins. Clustered throughout the carboxyl half of the protein are a series of serine-arginine (SR) repeats that are a characteristic feature of many RNA splicing factors. Antibodies raised against matrin CYP recognize a 106-kDa antigen that is detected in isolated nuclei and quantitatively subfractionates in the nuclear matrix. Laser scanning confocal microscopy localizes most of the anti-matrin CYP-specific antigen within the nucleus in a pattern of large bright speckles that co-localize with splicing factors and diffuse nucleoplasmic staining. A strikingly similar pattern of staining is observed in cells extracted for in situ nuclear matrices. A fusion protein containing the cyclophilin domain of matrin CYP exhibits cyclosporin A (CsA)-sensitive, peptidylprolyl cis-trans-isomerase activity that is characteristic of native cyclophilins. Although total rat liver nuclei contains predominantly CsA-resistant PPIase activity, the corresponding activity in the nuclear matrix is largely CsA-sensitive.The many functions associated with the cell nucleus are temporally and spatially regulated. Nuclear proteins and nucleic acids are often partitioned into functional domains (1). Examples include the nucleoli, heterochromatin, DNA replication sites (2, 3), and transcription domains (4, 5). In addition, a number of other domains have been identified that may also perform specific functions, such as the nuclear speckles (1, 6) and coiled (7) and promyelocytic leukemia (8 -10) bodies. Determining which molecules comprise these structures and how these molecules interact will lead to a greater understanding of the mechanisms responsible for their diverse functions and the role of nuclear architecture in these processes.The proteins and nucleic acids in the cell nucleus that resist solubilization by high salt extraction, nuclease digestion, and detergent solubilization constitute a structure termed the nuclear matrix (11,12). The isolated nuclear matrix has been shown to maintain a structure similar to and containing many of the functional properties associated with the nucleus (13).Several major proteins of the internal nuclear matrix have been identified by immunological methods and termed the nuclear matrins (14). The nuclear matrins include previously characterized proteins such as human RNP 1 A, human nRNP B (15), the nucleolar protein, B23/numatrin (14, 16), the hyperphosphorylated form of RNA pol II LS (17, 18), numerous SR-related proteins (19), and a 125-kDa acidic protein, termed matrin 3 (20). A recent study has confirmed the RNP nature of many of the major nuclear matrix proteins (21).Here we present results on the isolation of a cDNA that encodes an 88-kDa protein with a cyclophilin domain at the amino ter...

Research in our laboratory employs a dual approach to correlating genomic function and regulation with nuclear architecture. In one series of projects we are identifying, cloning, and studying the molecular, genetic, and functional properties of the nuclear matrix proteins which comprise the three-dimensional nuclear architecture. In a second series, we are using molecular labeling, microscopic, and computer imaging approaches to visualize in three dimensions sites of genomic organization and function within the in situ nuclear architecture and to obtain precise structural and spatio-temporal information about the individual sites where genomic function and regulation occurs. Additionally, our group is investigating the possible arrangement of the functional sites into higher-order domains and their relationship to the overall architecture and regulation in the cell nucleus. Nuclear Matrix Proteins as a Basis for Cenomic Organization, Function, and RegulationWhile a plethora of functional properties are associated with the isolated nuclear matrix [Berezney, 1991; Berezney et al., 19951, our knowledge of the proteins which compose the nuclear matrix is still in its infancy. Comprehensive analysis of the individual proteins is, therefore, critical as a basis for correlating higher order structural organization in the nucleus with the associated genomic functions.Studies in our laboratory are emphasizing the major proteins of the nuclear matrix (termed nuclear matrins) which are common among mammalian cells [Nakayasu and Berezney, 1991; Belgrader et al., 1991al. Microsequencing, molecular suggested that a significant portion of the major nuclear matrix proteins correspond to known pre-mRNP proteins, transcriptional or RNA splicing factors [Mattern et al., 1996; S. Kim, X. Wei, and R. Berezney, unpublished findings].Additional proteins are involved in other regulatory properties or are newly identified proteins whose function remains to be resolved. Our research group is currently concentrating its efforts on three nuclear matrix-associated proteins termed matrin 3, matrin cyp, and matrin p250.Matrin 3 is an acidic nuclear matrix protein (125 kDa). Antibodies to this protein decorate the nuclear interior with a fibrogranular pattern typical of the nuclear matrins [Nakayasu and Berezney, 1991; Belgrader et al., 1991bl. The analysis of matrin 3 human and rat cDNA sequences indicates a high degree of conservation among mammals [Belgrader et al., 1991bl. The lack of homologies with other proteins or functional motifs gives few clues to possible function, although the presence of over 40 potential phosphorylation sites suggests the role of protein phosphorylation. As a prelude to studying matrin 3 function, we are investigating the genomic organization of the matrin 3 gene in the rat. Preliminary results suggest the presence of at least two closely related genes and the possibility of alternatively spliced transcripts [Mortillaro et al., 1993; Somanathan et al., 19951. Matrin cyp is a 106 kDa protein that is localize...

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