title: The trypanosomatid-specific N terminus of RPA2 is required for RNA polymerase I assembly, localization, and function.
abstract: African trypanosomes are the only organisms known to use RNA polymerase I (pol I) to transcribe protein-coding genes. These genes include VSG, which is essential for immune evasion and is transcribed from an extranucleolar expression site body (ESB). Several trypanosome pol I subunits vary compared to their homologues elsewhere, and the question arises as to how these variations relate to pol I function. A clear example is the N-terminal extension found on the second-largest subunit of pol I, RPA2. Here, we identify an essential role for this region. RPA2 truncation leads to nuclear exclusion and a growth defect which phenocopies single-allele knockout. The N terminus is not a general nuclear localization signal (NLS), however, and it fails to accumulate unrelated proteins in the nucleus. An ectopic NLS is sufficient to reinstate nuclear localization of truncated RPA2, but it does not restore function. Moreover, NLS-tagged, truncated RPA2 has a different subnuclear distribution to full-length protein and is unable to build stable pol I complexes. We conclude that the RPA2 N-terminal extension does not have a role exclusive to the expression of protein-coding genes, but it is essential for all pol I functions in trypanosomes because it directs trypanosomatid-specific interactions with RPA1.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors.forename: Jan-Peter
authors.initials: JP
authors.lastname: Daniels
authors.forename: Keith
authors.initials: K
authors.lastname: Gull
authors.forename: Bill
authors.initials: B
authors.lastname: Wickstead
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D019295:Computational Biology; D003470:Culture Media / Q000378:metabolism; D055786:Gene Knockout Techniques; D008856:Microscopy, Fluorescence; D046912:Multiprotein Complexes / Q000378:metabolism; D011401:Promoter Regions, Genetic; D055550:Protein Stability; D015800:Protozoan Proteins / Q000378:metabolism*; D012318:RNA Polymerase I / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
chemical_list: D003470:Culture Media; D046912:Multiprotein Complexes; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D012318:RNA Polymerase I