Blom7alpha is a novel heterogeneous nuclear ribonucleoprotein K homology domain protein involved in pre-mRNA splicing that interacts with SNEVPrp19-Pso4.
The removal of introns from pre-mRNA is performed by the spliceosome that stepwise assembles on the pre-mRNA before performing two catalytic steps. The spliceosome-associated CDC5L-SNEV(Prp19-Pso4) complex is implicated in activation of the second catalytic step of pre-mRNA splicing, and one of its members, SNEV(Prp19-Pso4), is also implicated in spliceosome assembly. To identify interaction partners of SNEVPrp19-Pso4, we have performed yeast two-hybrid screenings. Among the putative binding partners was a so far uncharacterized protein carrying two heterogeneous nuclear ribonucleoprotein K homology domains that we termed Blom7alpha. Blom7alpha is expressed in all tissues tested, and at least three splice variants exist. After confirming direct and physical interaction of SNEV and Blom7alpha, we investigated if it plays a functional role during pre-mRNA splicing. Indeed, Blom7alpha co-localizes and co-precipitates with splicing factors and pre-mRNA and is present in affinity-purified spliceosomes. More importantly, addition of Blom7alpha to HeLa nuclear extracts increased splicing activity in a dose-dependent manner. Furthermore, we tested if Blom7alpha influences splice site selection using two different minigene constructs. Indeed, both 5'- as well as 3'-site selection was altered upon Blom7alpha overexpression. Thus we suggest that Blom7alpha is a novel splicing factor of the K homology domain family that might be implicated in alternative splicing by helping to position the CDC5L-SNEV(Prp19-Pso4) complex at the splice sites.
Grillari
Johannes
J
Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Vienna A-1190, Austria. johannes.grillari@boku.ac.at
Löscher
Marlies
M
Denegri
Marco
M
Lee
Kiseok
K
Fortschegger
Klaus
K
Eisenhaber
Frank
F
Ajuh
Paul
P
Lamond
Angus I
AI
Katinger
Hermann
H
Grillari-Voglauer
Regina
R
D017398:Alternative Splicing; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D034641:Heterogeneous-Nuclear Ribonucleoprotein K / Q000737:chemistry*; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000737:chemistry*; D006801:Humans; D007438:Introns; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012322:RNA Precursors / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D020798:Two-Hybrid System Techniques
D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C000590914:KHDC4 protein, human; D012322:RNA Precursors