title: Dynein Light Chain LC8 Is Required for RNA Polymerase I-Mediated Transcription in Trypanosoma brucei, Facilitating Assembly and Promoter Binding of Class I Transcription Factor A.
abstract: Dynein light chain LC8 is highly conserved among eukaryotes and has both dynein-dependent and dynein-independent functions. Interestingly, LC8 was identified as a subunit of the class I transcription factor A (CITFA), which is essential for transcription by RNA polymerase I (Pol I) in the parasite Trypanosoma brucei. Given that LC8 has never been identified with a basal transcription factor and that T. brucei relies on RNA Pol I for expressing the variant surface glycoprotein (VSG), the key protein in antigenic variation, we investigated the CITFA-specific role of LC8. Depletion of LC8 from mammalian-infective bloodstream trypanosomes affected cell cycle progression, reduced the abundances of rRNA and VSG mRNA, and resulted in rapid cell death. Sedimentation analysis, coimmunoprecipitation of recombinant proteins, and bioinformatic analysis revealed an LC8 binding site near the N terminus of the subunit CITFA2. Mutation of this site prevented the formation of a CITFA2-LC8 heterotetramer and, in vivo, was lethal, affecting assembly of a functional CITFA complex. Gel shift assays and UV cross-linking experiments identified CITFA2 as a promoter-binding CITFA subunit. Accordingly, silencing of LC8 or CITFA2 resulted in a loss of CITFA from RNA Pol I promoters. Hence, we discovered an LC8 interaction that, unprecedentedly, has a basal function in transcription.
authors.affiliation: Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Justin K
authors.initials: JK
authors.lastname: Kirkham
authors.affiliation: Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Sung Hee
authors.initials: SH
authors.lastname: Park
authors.affiliation: Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Tu N
authors.initials: TN
authors.lastname: Nguyen
authors.affiliation: Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Ju Huck
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA gunzl@uchc.edu.
authors.forename: Arthur
authors.initials: A
authors.lastname: Günzl
mesh_terms: D000818:Animals; D056445:Cytoplasmic Dyneins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012318:RNA Polymerase I / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D014157:Transcription Factors; D012318:RNA Polymerase I; C486069:Dynll1 protein, rat; D056445:Cytoplasmic Dyneins