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RNA interference in Trypanosoma brucei: Cloning of small interfering RNAs provides evidence for retroposon-derived 24–26-nucleotide RNAs

Published online by Cambridge University Press:  11 January 2002

APPOLINAIRE DJIKENG
Affiliation:
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8022, USA
HUAFANG SHI
Affiliation:
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8022, USA
CHRISTIAN TSCHUDI
Affiliation:
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8022, USA
ELISABETTA ULLU
Affiliation:
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8022, USA Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8022, USA
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Abstract

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.

Type
Research Article
Information
RNA , Volume 7 , Issue 11 , November 2001 , pp. 1522 - 1530
Copyright
© 2001 RNA Society

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RNA interference in Trypanosoma brucei: Cloning of small interfering RNAs provides evidence for retroposon-derived 24–26-nucleotide RNAs
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RNA interference in Trypanosoma brucei: Cloning of small interfering RNAs provides evidence for retroposon-derived 24–26-nucleotide RNAs
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