Insights into a CRM1-mediated RNA-nuclear export pathway in Trypanosoma cruzi.
Nuclear export and import of proteins and RNAs is a regulated process that permits the control of protein expression during cell development and differentiation. In all eukaryotic organisms transport of proteins to specific cellular compartments requires specific signaling sequences. Proteins that shuttle between nucleus and cytoplasm bear nuclear localization signals (NLS) and/or nuclear export signals (NES) and some of them can carry mRNAs, as part of shuttling ribonucleoprotein complexes. In this work we describe in the protozoan parasite Trypanosoma cruzi, a CRM1/exportin1 nuclear export factor named TcCRM1. This protein contains the conserved central region (CCR) that interacts with NES sequences present within cargo molecules, and the Cys residue involved in covalent binding to the Streptomyces metabolite leptomycin B (LMB). By subcellular fractionation we show that TcCRM1, a protein of about 117 kDa, has nuclear localization. We also demonstrate that LMB inhibits the replication of T. cruzi in a dose-dependent manner. In situ hybridization experiments performed with a Texas red-coupled oligo(dT) probe revealed that LMB produced a partial short-term accumulation of a poly(A)+RNA subset in the nucleus. Some mRNAs such as HSP70, TcUBP2/1 and TcPABP1 are reduced or disappeared from the cytoplasm of LMB treated cells. In sharp contrast with metazoans, no effect was observed on two U snRNAs subcellular localization, implying that a different export route might exist for these RNAs in trypanosomes.
Cuevas
Ileana C
IC
Instituto de Investigaciones Biotecnológicas, Instituto Tecnológico de Chascomús, CONICET-UNSAM, 1650 San Martín, Provincia de Buenos Aires, Argentina.
Frasch
Alberto C C
AC
D'Orso
Iván
I
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D000981:Antiprotozoal Agents / Q000378:metabolism / Q000494:pharmacology; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D016054:DNA, Protozoan / Q000737:chemistry; D005231:Fatty Acids, Unsaturated / Q000378:metabolism / Q000494:pharmacology; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011061:Poly A / Q000378:metabolism; D039122:Poly(A)-Binding Protein I / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D034443:RNA Transport; D012333:RNA, Messenger / Q000378:metabolism; D016053:RNA, Protozoan / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014349:Trypanosoma cruzi / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000097606:Exportin 1 Protein
D000981:Antiprotozoal Agents; D016054:DNA, Protozoan; D005231:Fatty Acids, Unsaturated; D018840:HSP70 Heat-Shock Proteins; D028884:Karyopherins; D011061:Poly A; D039122:Poly(A)-Binding Protein I; D012333:RNA, Messenger; D016053:RNA, Protozoan; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein; C038753:leptomycin B