A New, Expressed Multigene Family Containing a Hot Spot for Insertion of Retroelements Is Associated with Polymorphic Subtelomeric Regions of Trypanosoma brucei

Abstract

We describe a novel gene family that forms clusters in subtelomeric regions of Trypanosoma brucei chromosomes and partially accounts for the observed clustering of retrotransposons. The ingi and ribosomal inserted mobile element (RIME) non-LTR retrotransposons share 250 bp at both extremities and are the most abundant putatively mobile elements, with about 500 copies per haploid genome. From cDNA clones and subsequently in the T. brucei genomic DNA databases, we identified 52 homologous gene and pseudogene sequences, 16 of which contain a RIME and/or ingi retrotransposon inserted at exactly the same relative position. Here these genes are called the RHS family, for retrotransposon hot spot. Comparison of the protein sequences encoded by RHS genes (21 copies) and pseudogenes (24 copies) revealed a conserved central region containing an ATP/GTP-binding motif and the RIME/ingi insertion site. The RHS proteins share between 13 and 96% identity, and six subfamilies, RHS1 to RHS6, can be defined on the basis of their divergent C-terminal domains. Immunofluorescence and Western blot analyses using RHS subfamily-specific immune sera show that RHS proteins are constitutively expressed and occur mainly in the nucleus. Analysis of Genome Survey Sequence databases indicated that the Trypanosoma brucei diploid genome contains about 280 RHS (pseudo)-genes. Among the 52 identified RHS (pseudo)genes, 48 copies are in three RHS clusters located in subtelomeric regions of chromosomes Ia and II and adjacent to the active bloodstream form expression site in T. brucei strain TREU927/4 GUTat10.1. RHS genes comprise the remaining sequence of the size-polymorphic "repetitive region" described for T. brucei chromosome I, and a homologous gene family is present in the Trypanosoma cruzi genome.African trypanosomes, including Trypanosoma brucei, are unicellular protists that are responsible for diseases affecting humans and livestock. The nuclear chromosomes of T. brucei can be grouped into three size classes based on their migration in pulsed-field gel electrophoresis: 11 pairs of diploid megabase chromosomes (1 to 6 Mb) that contain the housekeeping genes and represent about 80% of the nuclear DNA content, a few intermediate-sized chromosomes (200 to 900 kb), and an undetermined number of minichromosomes (in the range of 100 that were 50 to 150 kb in size) (27,44,68). The intermediate and minichromosomes, whose ploidy is uncertain, play a role in antigenic variation. The genome sizes of different T. brucei isolates can vary by up to 30% (30,32,44,45). T. brucei TREU927/4 GUTat10.1, the reference strain for genome sequencing, contains an estimated 62-Mb diploid nuclear genome, including 53.4 Mb of diploid megabase chromosomal DNA (44).T. brucei has a life cycle that alternates between the tsetse fly and the mammal. In the bloodstream of their mammalian hosts, the parasites evade the immune response by antigenic variation, a continual switching of the variant surface glycoprotein (VSG) that constitutes the surface coat. Although each blood...

Comparative transcriptome analysis of small noncoding RNAs in different stages of Trypanosoma brucei

“…The RHS family includes six genes, RHS1 to RHS6, defined by their divergent C-terminal domains (Bringaud et al 2002). In the slender form, most siRNAs were found to target RHS3, followed by RHS4, RHS1, and RHS5 (Fig.…”

“…Based on the analysis for the targets of siRNA (Supplemental Tables S5, S6), our results further indicate that both RHS and VSG families are potentially regulated by siRNAs. Evidence suggests that the RHS family consists of at least six different RHS genes and a variable number of pseudogenes (Bringaud et al 2002). In addition, it has been reported that the absence of AGO, a key enzyme in the RNAi pathway, led to an increase of steady-state transcripts of RHS genes and pseudogenes (Durand-Dubief et al 2007).…”

Hemizygous subtelomeres of an African trypanosome chromosome may account for over 75% of chromosome length

“…In the left subtelomeric region of chrI, most of the genes belong to the novel multigene family called RHS (Bringaud et al 2002). Little is known about RHS proteins except that they are constitutively expressed and localize in or around the nucleus.…”

“…Among these are rapidly evolving gene families such as RHS, highly variable genes of unknown function that contain a hot spot of insertion for retroelements (Bringaud et al 2002), LRRP (Leucine Rich Repeat Protein), and VSGs.…”

The transcriptomes of Trypanosoma brucei Lister 427 and TREU927 bloodstream and procyclic trypomastigotes

“…The VSGs of T. brucei are expressed from telomeric expression sites containing up to 10 additional co-transcribed genes called "expression-siteassociated" genes (ESAGs) [15]. In addition to the ESAGs and VSGs at the telomeres, the T. brucei genome contains a large number of chromosome-internal or sub-telomeric ESAG and VSG genes and pseudogenes, often alternating with repetitive elements [16][17][18]. Transcripts from the telomeric expression sites in the bloodstream-form are abundant, and will cross-hybridise with similar genes and pseudogenes from elsewhere in the genome.…”