The Argonaute protein TbAGO1 contributes to large and mini-chromosome segregation and is required for control of RIME retroposons and RHS pseudogene-associated transcripts.
The protist Trypanosoma brucei possesses a single Argonaute gene called TbAGO1 that is necessary for RNAi silencing. We previously showed that in strain 427, TbAGO1 knock-out leads to a slow growth phenotype and to chromosome segregation defects. Here we report that the slow growth phenotype is linked to defects in segregation of both large and mini-chromosome populations, with large chromosomes being the most affected. These phenotypes are completely reversed upon inducible re-expression of TbAGO1 fused to GFP, demonstrating their link with TbAGO1. Trypanosomes that do not express TbAGO1 show a general increase in the abundance of transcripts derived from the short retroposon RIME (Ribosomal Interspersed Mobile Element). Supplementary large RIME transcripts emerge in the absence of RNAi, a phenomenon coupled to the disappearance of short transcripts. These fluctuations are reversed by inducible expression of GFP::TbAGO1. Furthermore, we use a combination of Northern blots, RT-PCR and sequencing to reveal that RNAi controls expression of transcripts derived from RHS (Retrotransposon Hot Spot) pseudogenes (RHS genes with retro-element(s) integrated within their coding sequence). Absence of RNAi also leads to an increase of steady-state transcripts from regular RHS genes (those without retro-element), indicating a role for pseudogene in control of gene expression. However, analysis of retroposon abundance and arrangement in the genome of multiple clonal cell lines of TbAGO1-/- failed to reveal movement of mobile elements despite the increased amounts of retroposon transcripts.
Durand-Dubief
Mickaël
M
Régulation et Dynamique des Génomes, Muséum National d'Histoire Naturelle, INSERM & CNRS, 75005 Paris, France.
Absalon
Sabrina
S
Menzer
Linda
L
Ngwabyt
Sandra
S
Ersfeld
Klaus
K
Bastin
Philippe
P
D000818:Animals; D060565:Argonaute Proteins; D020090:Chromosome Segregation; D017353:Gene Deletion; D020868:Gene Silencing; D005816:Genetic Complementation Test; D011544:Pseudogenes / Q000235:genetics*; D016053:RNA, Protozoan / Q000096:biosynthesis; D016601:RNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D018626:Retroelements / Q000235:genetics*; D014158:Transcription, Genetic / Q000502:physiology*; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000254:growth & development / Q000502:physiology*