McCracken S et al. (2005) J Biol Chem "Proteomic analysis of SRm160-containing complexes reveals a conserved association with cohesin."

Moens P, Downey M, Longman D, McCracken S, Blencowe BJ, Jessberger R, Marcon E, Wilde A, Nickerson JA, Emili A, Caceres JF

[J Biol Chem, 2005]

In this report we describe a rapid immunoaffinity purification procedure for gel-free tandem mass spectrometry-based analysis of endogenous protein complexes, and apply it to the characterization of complexes containing the SRm160 (serine/arginine-repeat related nuclear matrix protein of 160kDa) splicing coactivator. In addition to promoting splicing, SRm160 stimulates 3 -end processing via its N-terminal PWI nucleic acid binding domain, and is found in a post-splicing exon junction complex that has been implicated in coupling splicing to mRNA turnover, export and translation. Consistent with these known functional associations, we find that the majority of proteins identified in SRm160-containing complexes are associated with pre-mRNA processing. Interestingly, SRm160 is also associated with factors involved in chromatin regulation and sister chromatid cohesion, specifically the cohesin subunits, SMC1alpha, SMC3, hRAD21 and SA2. Gradient fractionation suggests that there are two predominant SRm160-containing complexes, one enriched in splicing components and the other enriched in cohesin subunits. Co-immunoprecipitation and colocalization experiments, as well as combinatorial RNA interference in C. elegans, support the existence of conserved and functional interactions between SRm160 and cohesin.

McCracken S et al. (2003) J Biol Chem "An evolutionarily conserved role for SRm160 in 3'-end processing that functions independently of exon junction complex formation."

Blencowe BJ, Longman D, McCracken S, Johnstone IL, Caceres JF

[J Biol Chem, 2003]

SRm160 (the SR-related nuclear matrix protein of 160 kDa) functions as a splicing coactivator and 3'-end cleavage-stimulatory factor. It is also a component of the splicing-dependent exon-junction complex (EJC), which has been implicated in coupling of pre-mRNA splicing with mRNA turnover and mRNA export. We have investigated whether the association of SRm160 with the EJC is important for efficient 3'-end cleavage. The EJC components RNPS1, REF, UAP56, and Y14 interact with SRm160. However, when these factors were tethered to transcripts, only SRm160 and RNPS1 stimulated 3'-end cleavage. Whereas SRm160 stimulated cleavage to a similar extent in the presence or absence of an active intron, stimulation of 3'-end cleavage by tethered RNPS1 is dependent on an active intron. Assembly of an EJC adjacent to the cleavage and polyadenylation signal in vitro did not significantly affect cleavage efficiency. These results suggest that SRm160 stimulates cleavage independently of its association with EJC components and that the cleavage-stimulatory activity of RNPS1 may be an indirect consequence of its ability to stimulate splicing. Using RNA interference (RNAi) in Caenorhabditis elegans, we determined whether interactions between SRm160 and the cleavage machinery are important in a whole organism context. Simultaneous RNAi of SRm160 and the cleavage factor CstF-50 (Cleavage stimulation factor 50-kDa subunit) resulted in late embryonic developmental arrest. In contrast, RNAi of CstF-50 in combination with RNPS1 or REFs did not result in an apparent phenotype. Our combined results provide evidence for an evolutionarily conserved interaction between SRm160 and the 3'-end cleavage machinery that functions independently of EJC

sulfiredoxin activity

Catalysis of the reaction: peroxiredoxin-(S-hydroxy-S-oxocysteine) + ATP + 2 R-SH = peroxiredoxin-(S-hydroxycysteine) + ADP + phosphate + R-S-S-R.

eriodictyol 3'-O-methyltransferase activity

Catalysis of the reaction: (S)-eriodictyol + S-adenosyl-L-methionine = (S)-homoeriodictyol + H+ + S-adenosyl-L-homocysteine.

(S)-coclaurine-N-methyltransferase activity

Catalysis of the reaction: S-adenosyl-L-methionine + (S)-coclaurine = S-adenosyl-L-homocysteine + (S)-N-methylcoclaurine.

obsolete glutathione oxidoreductase activity

OBSOLETE. Catalysis of the reaction: protein-S-S-glutathione + glutathione-SH = protein-SH + glutathione-S-S-glutathione.

(S)-scoulerine 9-O-methyltransferase activity

Catalysis of the reaction: (S)-scoulerine + S-adenosyl-L-methionine(1+) = (S)-tetrahydrocolumbamine + S-adenosyl-L-homocysteine + H+.

3'-hydroxy-N-methyl-(S)-coclaurine 4'-O-methyltransferase activity

Catalysis of the reaction: S-adenosyl-L-methionine + 3'-hydroxy-N-methyl-(S)-coclaurine = S-adenosyl-L-homocysteine + (S)-reticuline.

(S)-tetrahydroprotoberberine N-methyltransferase activity

Catalysis of the reaction: S-adenosyl-L-methionine + (S)-7,8,13,14-tetrahydroprotoberberine = S-adenosyl-L-homocysteine + cis-N-methyl-(S)-7,8,13,14-tetrahydroprotoberberine.

(R,S)-reticuline 7-O-methyltransferase activity

Catalysis of the reaction: (S)- or (R)-reticuline + S-adenosyl-L-methionine = (S)- or (R)-laudanine + H+ + S-adenosyl-L-homocysteine.