The small RNA repertoire of Dictyostelium discoideum and its regulation by components of the RNAi pathway
Andrea Hinas
2
Johan Reimega rd
1
E. Gerhart H. Wagner
1
Wolfgang Nellen
0
Victor R. Ambros
3
Fredrik So derbom
2
0
Department of Genetics, Kassel University
, Heinrich Plett Strasse 40, 34132 Kassel,
Germany
1
Department of Cell and Molecular Biology, Biomedical Center, Uppsala University
, Box 596, SE-75124 Uppsala,
Sweden
2
Department of Molecular Biology, Biomedical Center, Swedish University of Agricultural Sciences
, Box 590, SE-75124 Uppsala,
Sweden
3
Department of Genetics, Dartmouth Medical School
, Hanover,
NH 03755, USA
Small RNAs play crucial roles in regulation of gene expression in many eukaryotes. Here, we report the cloning and characterization of 18-26 nt RNAs in the social amoeba Dictyostelium discoideum. This survey uncovered developmentally regulated microRNA candidates whose biogenesis, at least in one case, is dependent on a Dicer homolog, DrnB. Furthermore, we identified a large number of 21 nt RNAs originating from the DIRS-1 retrotransposon, clusters of which have been suggested to constitute centromeres. Small RNAs from another retrotransposon, Skipper, were significantly up-regulated in strains depleted of the second Dicer-like protein, DrnA, and a putative RNA-dependent RNA polymerase, RrpC. In contrast, the expression of DIRS-1 small RNAs was not altered in any of the analyzed strains. This suggests the presence of multiple RNAi pathways in D. discoideum. In addition, we isolated several small RNAs with antisense complementarity to mRNAs. Three of these mRNAs are developmentally regulated. Interestingly, all three corresponding genes express longer antisense RNAs from which the small RNAs may originate. In at least one case, the longer antisense RNA is complementary to the spliced but not the unspliced pre-mRNA, indicating synthesis by an RNA-dependent RNA polymerase.
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Since their initial discovery in worms, 1826 nt small
RNAs have now been identified in many eukaryotes (1,2).
By antisense complementarity, they confer specificity to
associated protein complexes and can thereby regulate
expression of their target genes at the level of
transcription, mRNA stability or translation (35). These small
RNAs can be divided into two main classes; microRNAs
(miRNAs), which are processed from imperfectly
basepaired hairpin transcripts, and small interfering RNAs
(siRNAs), which are derived from long double-stranded
RNAs (dsRNAs) (57). The dsRNA precursors of
siRNAs originate e.g. from viruses, repetitive elements
or are synthesized by an RNA-dependent RNA
polymerase (RdRP) using a single-stranded RNA as a template
(2,810). Despite their different origin, miRNA and
siRNA pathways share many similarities. In both cases,
these small RNAs are processed from precursors into
mature small RNAs by the RNase III-type Dicer proteins,
and are subsequently incorporated in an effector complex
that contains an ArgonautePiwi family protein (5,6). The
miRNA/siRNA guides the protein complex to its
complementary target RNA and induces cleavage of the target
if the small RNA and target RNA form a perfectly or
close to perfectly base-paired duplex. In plants, most
miRNAs exert their effect in this way. If base pairing is
only partial, as is the common feature of animal miRNA
target interactions, the main effect seems to be inhibition
of translation, although some mRNA degradation is also
frequently observed (4).
miRNAs are common in multicellular organisms and
animal viruses and were only recently discovered in a
unicellular organism, the green alga Chlamydomonas
reinhardtii (6,1113). In animals and plants, miRNAs play
important cellular roles by modulating the expression of
endogenous genes and it has been estimated that up to
onethird of all human genes may be regulated by miRNAs
(5,14). The physiological role of miRNAs in C. reinhardtii
is still not known, however, verified targets include genes
encoding flagellum-associated proteins (11,13).
siRNAs are present in eukaryotes from all major
phylogenetic branches including plants, animals and
fungi where they act e.g. in a defense mechanism against
viral RNAs and mobilization of transposons (2,15).
Repetitive elements, such as transposons and
retrotransposons, constitute substantial parts of the centromeres
in many eukaryotes, and the RNAi machinery is required
for silencing of centromeric repeats in e.g. fission yeast
(16,17). Although most studies on natural siRNAs have
focused on their roles in defense against viruses and
repetitive elements, an increasing number of reports point
to an additional role in regulation of non-transposon
genes. For example, large-scale cloning of small RNAs
from Caenorhabditis elegans and Arabidopsis thaliana has
identified many small RNAs with antisense
complementarity to genes other than repetitive elements (1820).
Recently, such small RNAs were demonstrated to be
involved in regulation of overlapping A. thaliana genes
during salt stress and bacterial infection (20,21).
Furthermore, whole-genome microarray analyses and (...truncated)