Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.31.1 (micrococcal nuclease)
 2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have identified six distinct factors necessary for pre-mRNA splicing in vitro by selective inactivation and complementation studies, and by fractionation procedures. Splicing factor 1 (SF1) is sensitive to micrococcal nuclease, and appears to consist of at least U1 and U2 snRNPs, since splicing is inhibited when the 5' termini of U1 and U2 snRNAs are removed by site-directed cleavage with RNAase H. SF2 is a micrococcal nuclease-resistant factor present in the nuclear extract but absent from an S100 extract. SF3 is a factor that can be preferentially inactivated by moderate heat treatment. Two additional factors (SF4A and SF4B) were identified by fractionation of the nuclear extract using spermine-agarose and CM-sepharose chromatography. SF1, SF2, and SF4B appear to be required for cleavage of the pre-mRNA at the 5' splice site and lariat formation, whereas SF3 and SF4A are only required for cleavage at the 3' splice site and exon ligation.
 ...
PMID:Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for pre-mRNA splicing in vitro. 299 74

We have used a complementation assay to test for activities required for the splicing of pre-mRNA in vitro. During the hypotonic lysis of HeLa cells, two components are released from the nuclei that specifically stimulate splicing in an extract prepared from washed nuclei. The two activities separate during chromatography on DEAE-Sepharose. One of these activities [splicing factor (SF)2] co-purified through several steps with the lariat debranching enzyme and with a nuclease which degrades the linear portion of lariat RNAs. These enzymes could, however, be separated from SF2 by chromatography on heparin-Sepharose. SF2 fractionates as a single protein with an apparent mol. wt. of 50 000. SF2 is resistant to mild heat treatment and to treatment with micrococcal nuclease, but it is inactivated by N-ethylmaleimide, suggesting that it is a protein which is not associated with an essential RNA component. When SF2 is absent in a complementation assay, the generation of both intermediates and final products of the splicing reaction is completely abolished. Thus, SF2 functions in an early step of the splicing process.
 ...
PMID:Purification of a protein required for the splicing of pre-mRNA and its separation from the lariat debranching enzyme. 409 89

Alternative splicing of the adenovirus-2 E1A pre-mRNA involves the use of three 5' splice sites and is modulated during infection because the 13S mRNA and 9S mRNA reactions are predominant during the early and late periods, respectively. We had previously reproduced in vitro the 13S to 9S modulation with nuclear extracts isolated from infected HeLa cells and shown that high molecular weight viral RNAs are involved in this modulation, most likely by sequestering or titrating general splicing factors. To further test this hypothesis, we titrated splicing factors from an uninfected nuclear extract using competitor RNA or by progressive inactivation of splicing factors with monoclonal antibodies. We found that the 13S to 9S modulation occurs when titrating only with certain RNAs (essentially adenoviral RNAs), and also by progressively inactivating the 9G8 SR splicing factor. The demonstration that late nuclear extracts contain levels of active SR splicing factors limiting for the 13S reaction has been made by complementation experiments. We show that late nuclear extracts do not complement SR factor-deficient extracts, whereas late extracts treated with micrococcal nuclease complement them. Furthermore, complementation of late nuclear extracts with each of the three 30-35-kDa SR factors (9G8, SC35, and SF2/ASF) restores an efficient 13S mRNA reaction. Thus, our results provide evidence that the 13S to 9S modulation is triggered through a titration of SR factors required for the 13S mRNA reaction by major late transcripts that accumulate in nuclei late in infection.
 ...
PMID:Titration of serine/arginine (SR) splicing factors during adenoviral infection modulates E1A pre-mRNA alternative splicing. 749 25