Evidence that a nuclear matrix protein participates in premessenger RNA splicing

Specific apolipoprotein B (apoB) mRNA editing can be performed in vitro on apoB RNA substrates. Native gels and glycerol gradient sedimentation have been used to determine the physical properties of the in vitro editing activity in rat liver cytosolic S100 extracts. ApoB RNA substrates were progressively assembled as 27S complexes for 3 hr with similar kinetics as seen for the accumulation of edited RNA. Assembly was not observed on RNAs from apoB deletion constructs that did not support editing. The 27S complex contained both edited and unedited RNA sequences. Inhibition of 27S complex assembly by vanadyl-ribonucleoside complexes was accompanied by inhibition of editing. Based on these data, we propose that the 27S complex is the in vitro "editosome." A "mooring sequence" model for RNA recognition and editosome assembly has been proposed involving RNA sequences flanking the edited nucleotide.

Section: Methodsmentioning

confidence: 99%

In vitro apolipoprotein B mRNA editing: identification of a 27S editing complex.

Smith

Kuo

Backus

et al. 1991

“…Recent microscopic studies have suggested that pre-mRNAs are both processed and transported in discrete nuclear "tracks" that might correspond to nuclear filaments (8)(9)(10)(11). In addition, earlier experiments revealed a preferential association of pre-mRNA with the nuclear matrix (12)(13)(14)(15)(16). Strong evidence for the relationship of the nucleoskeleton to gene expression, however, has been lacking due to the phenomenological nature of biochemical matrix preparations.…”

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confidence: 99%

“…32P-labeled splicing precursor RNA was transcribed in vitro from the MINX adenovirus minigene containing a single intron (16). Precursor RNA was capped during synthesis.…”

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confidence: 99%

Nuclear-mitotic apparatus protein: a structuralprotein interface between the nucleoskeleton and RNA splicing.

Zeng

Berget

et al. 1994

Vertebrate splicing factors are localized to discrete domains within the nuclei of somatic cells. The mechanism whereby such nuclear domains, identified as speckles by immunofluorescence microscopy, are generated is unclear. Recent studies suggest that the spatial order within the nucleus is maintained by nuclear matrix factors. Here we show that a protein in the nuclear matrix and mitotic apparatus [nuclearmitotic apparatus protein, NuMA; Lydersen, B. & Pettijohn, D. (1980) Cell 22, 489-499] colocalizes with splicing factors in interphase nuclei and is associated with small nuclear ribonucleoproteins in a complex immunoprecipitated from HeLa extract with small nuclear ribonucleoprotein antibodies. Moreover, NuMA associates with splicing complexes that are reconstituted in vitro using wild-type pre-mRNA, but not with nonspecific RNA. Cumulatively, these observations suggest a function of NuMA or NuMA-like proteins in interphase cells in providing a bridge between RNA processing and the nucleoskeleton.native splicing (23), although no correlation has been made between the isoforms and multiple functions for NuMA. In mitotic cells, NuMA appears to play a role in spindle microtubule formation and post-mitotic nuclear assembly as shown by various studies on microtubule inhibition, antibody injection, and expression of truncated NuMA proteins (18)(19)(20)(24)(25)(26)(27). Beyond its identity as a nuclear matrix protein (17,19), however, little is known of the function of NuMA in the interphase nucleus.Here we investigate the role of NuMA in interphase nuclei by the ability of NuMA-specific antibodies to stain discrete foci and to immunoprecipitate nuclear assemblies. Specifically, we show that NuMA colocalized with the small nuclear ribonucleoprotein particles (snRNPs) required for processing of pre-mRNAs in interphase. Furthermore, we show that NuMA associates in vitro with both nuclear snRNPs and reconstituted spliceosomes.The eukaryotic nucleus is a highly organized structure. Electron microscopy has provided excellent morphological evidence for the existence ofa complex nucleoskeleton (1, 2). An intermediate filament-like nuclear network termed core filaments has been revealed by a sequential extraction procedure (2, 3). These 9-to 13-nm filaments may be the core structure to which other proteins associate to form the nuclear matrix. Many nuclear activities, such as synthesis of DNA and RNA and RNA splicing, have been localized in discrete nuclear domains rather than diffusely distributed throughout the nucleoplasm (4-7). Particularly, the localizations of these specific domains persist after the removal of chromatin, indicating that the nuclear matrix has a key function in maintaining spatial order within the nucleus. Recent microscopic studies have suggested that pre-mRNAs are both processed and transported in discrete nuclear "tracks" that might correspond to nuclear filaments (8-11). In addition, earlier experiments revealed a preferential association of pre-mRNA with the nuclear matrix (12-16). Str...

“…However, it remains to be established how the nuclear matrix supports this organization of chromatin, and more importantly, the biochemical composition and sequence specificity of these anchorage sites are only beginning to be defined (6). The nuclear matrix has also been reported to have a role in mRNA transcription and processing via its involvement in attachment and/or association with newly transcribed mRNA (9), ribonucleoprotein particles (10), pre-mRNA splicing machinery (11,12), and steroid receptors (13,14). To attribute structure-function relationships to these reported associations, it is essential to characterize the nuclear matrix proteins involved and the nature of their interactions.…”

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confidence: 99%

Sequence-specific DNA-binding proteins are components of a nuclear matrix-attachment site.

Dworetzky

Wright

Fey

et al. 1992

122

We have identified a nuclear matrixattachment region within an upstream element of a human H4 histone gene promoter. Nuclear matrix proteins, isolated and solubilized from HeLa S3 cells, were found to interact with sequence specificity at this matrix-attachment region. Several types of assays for protein-DNA interaction showed that the minimal sequence for the nuclear matrix protein-DNA interaction was 5'-TGACGTCCATG-3'; the underlined region corresponds to the core consensus sequence for ATF transcription factor binding. Two proteins with molecular masses of 43 and 54 kDa were identified by UV-crosslinking analysis as integral components of this protein-DNA complex. The molecular masses of these proteins and the ATF-binding site consensus sequence suggest that these proteins are members of the ATF family. Our results provide direct evidence for nuclear matrix localization of sequence-specific DNA-binding factors for an actively transcribed gene. The proximity of a strong positive transcriptional regulatory element to the matrix-attachment region of this gene suggests that the nuclear matrix may serve to localize and concentrate trans-acting factors that facilitate regulation of gene expression.The functional diversity of the nuclear matrix is becoming increasingly apparent. The proteinaceous components are being better characterized, and evidence continues to accumulate for variations in nuclear matrix protein composition that reflect cell structure and function (1-3). The nuclear matrix, originally defined by Berezney and Coffey (4) as the insoluble skeletal framework within the nucleus, can maintain the structural integrity of the nucleus and provides anchor sites for DNA attachment (5, 6). These anchor sites have been reported to constrain DNA into loop structures of '60 kilobases (kb) (7,8). It is generally acknowledged that the three-dimensional conformation of chromatin can affect transcriptional regulation. However, it remains to be established how the nuclear matrix supports this organization of chromatin, and more importantly, the biochemical composition and sequence specificity of these anchorage sites are only beginning to be defined (6). The nuclear matrix has also been reported to have a role in mRNA transcription and processing via its involvement in attachment and/or association with newly transcribed mRNA (9), ribonucleoprotein particles (10), pre-mRNA splicing machinery (11,12), and steroid receptors (13,14). To attribute structure-function relationships to these reported associations, it is essential to characterize the nuclear matrix proteins involved and the nature of their interactions.The identification of regions of DNA attachment to the nuclear matrix [(matrix-attachment regions (MARs)] has been reported for several genes, including mouse immunoglobulin K chain (15), rat a2-macroglobulin (16), human interferon ( (17), several developmentally regulated genes of Drosophila melanogaster (18), human f3-globin (19), and the human apolipoprotein B gene (20). There are also data demonst...