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. Dynein light chain LC8 was originally discovered as a component of the outer arm axonemal dynein in Chlamydomonas reinhardtii (1) but was later found to be present also in cytoplasmic dyneins 1 and 2 (2-4). LC8 is conserved throughout eukaryotic genomes (5). As a part of the dynein motor, LC8 is important for fundamental cellular processes, such as tubulin minus-enddirected intracellular transport, chromatid separation during mitosis, and nuclear migration (6), as well as flagellum-specific functions, namely, motility, intraflagellar transport (7), and ciliogenesis (8, 9). While not essential in Saccharomyces cerevisiae (10), mutation or knockdown of LC8 is embryonic lethal in animals (8,9,11,12). Given that LC8 is more conserved between species than other components of the dynein motor and that LC8 is present in organisms which lack a dynein motor, it was likely that LC8 had nondynein functions (3, 5). LC8 has since been shown to interact with several different proteins and to affect various cellular processes, including protein localization and stability, transcription regulation, and apoptosis (13-15).At physiological pH, LC8 exists almost exclusively as a dimer (16, 17), interacting with partner proteins via two identical sites generated at the dimer interface which bind to diverse short, linear motifs (16,18,19). LC8 promotes the dimerization of its binding partners through aligning dimerization domains present in the partner protein (13,(20)(21)(22). While it was previously hypothesized that LC8 functions as a linker, allowing attachment of the dynein motor to its cargo, the emerging view is that interaction with LC8 induces dimerization, imparting new s...

The proteome and transcriptome of the infectious metacyclic form of Trypanosoma brucei define quiescent cells primed for mammalian invasion

“…5B). Both CITFA-1 and CITFA-2 have been shown to directly contact the VSG promoter in bloodstream VSG expression sites (BESs) (Kirkham et al, 2016) and CITFA-2 associates preferentially with the active BES VSG promoter (Nguyen et al, 2014).…”

Ebolaviruses: New roles for old proteins

“…VP35 also undergoes further protein–protein interactions that may affect viral genome transcription through the interaction with the cytoplasmic dynein light chain (LC8) [ 43 ]. LC8 is a highly conserved 8 kDa subunit of the cytoplasmic dynein motor complex but can also exist as a dimer in soluble form, which can affect viral transcription and assembly [ 44 , 45 ]. LC8 was seen to stabilise VP35 N-terminal oligomerisation in a dose-dependent manner and enhance viral genome synthesis [ 46 ].…”

May the Odds Be Ever in Your Favor: Non-deterministic Mechanisms Diversifying Cell Surface Molecule Expression

“…(D) RNA polymerase I transcribes polycistronic RNAs from active VSG expression sites. The CITFA transcription factor complex, which in T. brucei consists of CITFA subunits 1–7 (green circles) complexed with LC8/DYNLL1 (light green oval), is a basal transcription factor required for RNA pol I initiation ( Kirkham et al, 2016 ). The active VSG gene is typically transcribed last, preceded by expression-site associated genes (ESAGs) ( Pays et al, 2001 ; Hertz-Fowler et al, 2008 ).…”