Small interfering RNA-producing loci in the ancient parasitic eukaryote Trypanosoma brucei

Abstract

Background: At the core of the RNA interference (RNAi) pathway in Trypanosoma brucei is a single Argonaute protein, TbAGO1, with an established role in controlling retroposon and repeat transcripts. Recent evidence from higher eukaryotes suggests that a variety of genomic sequences with the potential to produce double-stranded RNA are sources for small interfering RNAs (siRNAs).

Results: To test whether such endogenous siRNAs are present in T. brucei and to probe the individual role of the two Dicer-like enzymes, we affinity purified TbAGO1 from wild-type procyclic trypanosomes, as well as from cells deficient in the cytoplasmic (TbDCL1) or nuclear (TbDCL2) Dicer, and subjected the bound RNAs to Illumina high-throughput sequencing. In wild-type cells the majority of reads originated from two classes of retroposons. We also considerably expanded the repertoire of trypanosome siRNAs to encompass a family of 147-bp satellite-like repeats, many of the regions where RNA polymerase II transcription converges, large inverted repeats and two pseudogenes. Production of these newly described siRNAs is strictly dependent on the nuclear DCL2. Notably, our data indicate that putative centromeric regions, excluding the CIR147 repeats, are not a significant source for endogenous siRNAs.

Conclusions: Our data suggest that endogenous RNAi targets may be as evolutionarily old as the mechanism itself.

Figure 1

Size distribution of analyzed T. brucei small RNAs in wild-type (wt), dcl1^−/−and dcl2^−/−cells.

Figure 2

Distribution of small RNAs aligning to retroposon elements. (A) ingi is a 5.25 kb element composed of an ingi-specific 4.75 kb fragment flanked by two separate halves of the RIME element family [28,29]. This element contains a single ORF (ORF1) flanked by two RIME elements. 2,326,864 reads in the wild-type library aligned to this element with 1,1321,119 reads from the sense and 1,194,745 reads from the antisense strand. Note that sense and antisense small RNA reads are shown separately. (B) The SLACS element is 6.8 kb long with two predicted ORFs. Sense (640,935 reads) and antisense (1,152,384 reads) small RNA reads are shown separately.

Figure 3

Small RNAs originating from two annotated pseudogenes, namely Tb09.160.3400 (A) and Tb927.5.290 (B). Sense and antisense reads are shown.

Figure 4

Long inverted repeats are a source of small RNAs. (A) Small RNA distribution on IR3. Reads were distributed randomly on the two repeats during alignment. An enlargement of the region in-between the two repeats is shown below. (B) Low-molecular weight RNAs isolated from various cell lines as indicated above each lane were separated by denaturing gel electrophoresis and analyzed by Northern hybridization with an IR3-specific probe. Hybridization to 5S rRNA was used as a loading control (load, bottom panel). See additional file 5 for sequences of the hybridization probes.

Figure 5

Validation of small RNA accumulation at CTU50. (A) Small RNA distribution. (B) Low-molecular weight RNAs isolated from various cell lines as indicated above each lane were separated by denaturing gel electrophoresis and analyzed by Northern hybridization with two oligonucleotides representing the most abundant reads. Loading control (load), hybridization to 5S rRNA. See additional file 5 for sequences of the hybridization probes.

Figure 6

Steady-state mRNA levels at two convergent transcription units in wild-type and DCL2KO cells. (A) and (B) Total RNA was prepared from wild-type and dcl2^−/− cells and analyzed by Northern hybridization with a probe specific for the genes indicated below the read distribution. See additional file 3 for sequences of the hybridization probes. The hybridization was quantitated by PhosphorImager analysis, normalized to the loading control (tRNA) and expressed as a ratio between dcl2^−/− and wt.