In animals and protozoa, gene-specific double-stranded RNA
(dsRNA) triggers degradation of homologous cellular RNAs, a
phenomenon known as RNA interference (RNAi). In vitro and in
vivo dsRNA is processed by a nuclease to produce 21–25-nt
small interfering RNAs (siRNAs) that guide target RNA degradation.
Here we show that activation of RNAi in Trypanosoma brucei
by expression or electroporation of actin dsRNA results in
production of actin siRNAs and that 10% of these RNAs sediment
as high-molecular-weight complexes at 100,000 × g.
To characterize actin siRNAs, we established a cloning and
enrichment strategy starting from 20–30 nt RNAs isolated
from high-speed pellet and supernatant fractions. Sequence analysis
revealed that actin siRNAs are 24–26 nt long and their
distribution relative to actin dsRNA was similar in the two
fractions. By sequencing over 1,300 fragments derived from the
high-speed pellet fraction RNA, we found abundant
24–26-nt-long fragments homologous to the ubiquitous
retroposon INGI and the site-specific retroposon SLACS. Northern
hybridization with strand-specific probes confirmed that
retroposon-derived 24–26-nt RNAs are present in both
supernatant and high-speed pellet fractions and that they are
constitutively expressed. We speculate that RNAi in trypanosomes
serves a housekeeping function and is likely to be involved
in silencing retroposon transcripts.