Biology Reference
In-Depth Information
2010
)], allowing a maximum of 2 bp mismatches in the reads. The resulting
sequence reads from 17
26 nt in length that matched the C. elegans genome were
considered as the total reads in the expression profiling. For examining the propor-
tion of all noncoding RNAs in the deep sequencing database, we aligned genome-
matched reads to the noncoding RNA list in WormBase using Blastn, with the
following parameters: -e 0.001-G 5-E 2-q-3-r 1-W 7-v 10-b 10.
1. Novel miRNA and 21U-RNA Discovery(
Bracht et al., 2010
)
For discovering novel miRNAs from the deep sequencing database, several soft-
ware tools based on different algorithms are publicly available, including miRDeep
(
Friedlander et al., 2008
), CID-miRNA (
Ty a g i et al.,2008
), miRank (
Xu et al., 2008
),
miRCat (
Moxon et al.,2008
), and miRanalyzer (
Hackenberg et al.,2009
). We have
chosen miRDeep, which uses a probabilistic model scoring both the compatibility of
the position and frequency of sequences with the model of miRNA biogenesis as well
as the stability of the characteristic hairpin structures of predicted pre-miRNAs. We
removed reads that match previously annotated noncoding RNAs and mRNAs using
Blastn and also removed reads that perfectly match known miRNAs and 21U-RNAs.
Then we loaded the remaining reads to miRDeep for searching for novel miRNAs and
used RNAfold to predict secondary structures of putative pre-miRNA hairpins
(
Hofacker, 2009
). For searching novel 21U-RNAs, we aligned the sequences of
21 nt in length with a 5
0
uracil in the remaining reads with the C. elegans genome
and judged by the previously reported characteristics of 21U-RNAs, that is, localiza-
tion on chromosomes and the upstream conserved motif (
Ruby et al.,2006
).
V. Analysis of miRNP Complexes
miRNAs function as part of the RISC protein complex (
Bartel, 2009
). Multiple
proteins also generally or specifically regulate miRNA biogenesis (
Kai and
Pasquinelli, 2010; Winter et al., 2009
). Protein immunoprecipitation followed by
MASS spectrometry has been done to identify proteins associated with RISC proteins
(
Zhang et al., 2007
). RNA immunoprecipitation (RIP) experiments have been used to
identify target mRNAs that associate with miRISC in vivo (
Zhang et al., 2007
). The
use of cross-linking to stabilize endogenous RNA-protein interactions followed by
immunoprecipitation of the protein complex of interest and isolation of the RNA
regions protected by the complex (cross-linking and immunoprecipitation, CLIP) has
enabled the identification of sequences specifically bound by miRISC (
Zisoulis et al.,
2010; Zisoulis et al., 2011
). Additionally, RIP allows the identification of the primary,
precursor, or mature miRNA that a particular protein or protein complex binds in vivo
(
Van Wynsberghe et al.,2011
). This method requires an antibody against the protein
or fusion-protein of interest. Parallel RIP analysis in a mutant worm strain automat-
ically provides a negative control for nonspecific antibody binding. Additionally, an
IgG antibody produced in the same species as the antibody of interest should be
included as a negative control to determine fold-enrichment. UV-cross-linking of live
worms allows endogenous RNA-protein interactions to be stabilized and avoids
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