Interaction of the Epstein-Barr virus mRNA export factor EB2 with human Spen proteins SHARP, OTT1, and a novel member of the family, OTT3, links Spen proteins with splicing regulation and mRNA export.
The Epstein-Barr virus early protein EB2 (also called BMLF1, Mta, or SM), a protein absolutely required for the production of infectious virions, shares properties with mRNA export factors. By using a yeast two-hybrid screen, we have identified the human protein OTT3 as an EB2-interacting factor. OTT3 is a new member of the Spen (split end) family of proteins (huSHARP, huOTT1, DmSpen, and muMINT), which are characterized by several N-terminal RNA recognition motifs and a highly conserved C-terminal SPOC (Spen Paralog and Ortholog C-terminal) domain that, in the case of SHARP, has been shown to interact with SMRT/NCoR corepressors. OTT3 is ubiquitously expressed as a 120-kDa protein. Transfected OTT3 is a nonshuttling nuclear protein that co-localizes with co-transfected EB2. We also showed that EB2 interacts with the SPOC domains of both OTT1 and SHARP proteins. Although the OTT3 interaction domain maps within the 40 N-terminal amino acids of EB2, OTT1 and SHARP interact within the C-terminal half of the protein. Furthermore, we demonstrated that the capacity of the OTT3 and OTT1 SPOC domains to interact with SMRT and repress transcription is far weaker than that of SHARP. Thus there is no evidence for a role of OTT3 in transcriptional regulation. Most interestingly, however, we have found that OTT3 has a role in splicing regulation; OTT3 represses accumulation of the alternatively spliced beta-thalassemia mRNAs, but it has no effect on the beta-globin constitutively spliced mRNA. Thus our results suggested a new function for Spen proteins related to mRNA export and splicing.
Hiriart
Edwige
E
INSERM U412, ENS-Lyon, IFR 128 Biosciences Lyon Gerland, Laboratoire de Virologie Humaine, 46 Allée d'Italie, 69364 Lyon, France.
Gruffat
Henri
H
Buisson
Monique
M
Mikaelian
Ivan
I
Keppler
Selina
S
Meresse
Patrick
P
Mercher
Thomas
T
Bernard
Olivier A
OA
Sergeant
Alain
A
Manet
Evelyne
E
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015514:Oncogene Proteins, Fusion; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D015514:Oncogene Proteins, Fusion; D010750:Phosphoproteins; C431574:RBM15 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C090236:SM protein, Human herpesvirus 4; C476510:SPEN protein, human; C402387:Spen protein, Drosophila; D015534:Trans-Activators; D014764:Viral Proteins