Slicer activity is indicated by the higher-mobility product ( fig. ( F) Northern blot of embryos to detect slicer cleavage of an injected GFP target mRNA with three complementary targets to miR-1 (3×PT-miR-1) in the presence (+) or absence (−) of miR-1 ( 7). Amino acid positions are based on the mammalian Ago2. The 90-nt deletion (Δ90) results in a predicted truncated protein lacking two of three catalytic residues. ( D) Total number of reads that matchmiR-451inwildtype, MZ dicer, and MZ ago2. ( C) Scheme of miR-144/miR-451 genomic loci and predicted secondary structure of both human and zebrafish pre-miR-451 (mature miRNA in red). Some miRNAs are shown as a reference for enhanced and reduced miRNAs (solid circles) miR-144-5′ (green) and miR-451-5′ (red) are expressed in the same pri-miRNA. ( A and B) Normalized reads from wild type versus MZ dicer (A) or MZ ago2 (B) libraries for all annotated zebrafish miRNAs. MicroRNA analysis in wild type (wt) and in MZ dicer and MZ ago2 mutants. These observations lead us to hypothesize that Ago2 slicer activity could participate in miRNA maturation ( fig. S1), a site where slicer activity cleaves the passenger strand in siRNAs ( 10– 12). The final templated base pairs with nucleotide 10 of the mature miRNA ( Fig. 1, C and D) and (iii) reads stopped at nucleotide 30, and longer reads carried one to five nontemplated uridines, with nucleotide 31 mostly being a non-templated U ( Fig. S5) with a 17-nt stem, whereas Dicer requires a >19-nt stem for efficient processing ( 9) (ii) miR-451 has a defined 5′ end but a variable 3′ end that extends over the loop region and ranges between 20 and 30 nt ( Fig. miR-451 differs from other “canonical” miRNAs for several reasons: (i) It is encoded in a conserved 42-nt hairpin ( fig. On the basis of read frequency, reproducibility, and evolutionary conservation, we focused subsequent analysis on miR-451. Several miRNAs appeared refractory to dicer loss of function, notably miR-451-5′, miR-2190-5′, miR-2190-3′, and miR-735-5′ ( Fig. Of the ~2 million reads per sample, 69 to 82% mapped to known 5′- or 3′-derived miRNAs in the wild type, whereas 4 to 9% mapped to miRNAs in the MZ dicer mutants ( fig. We analyzed 48-hour-old embryos in two wild-type replicates and two dicer mutant alleles ( 8), dicer hu715 and dicer hu896 ( fig. But are there functional miRNAs that bypass Dicer? To identify pathways that might process miRNAs in a Dicer-independent manner, we sequenced small RNAs (19 to 36 nt) in wild-type and maternal-zygotic dicer mutants (MZ dicer)( 7). In contrast to Drosha, Dicer has been viewed as a central processing enzyme in the maturation of small RNAs ( 2). Recent studies have identified several miRNA classes that bypass Drosha-mediated processing, namely miRtrons, tRNAZ, and small nucleolar RNA (snoRNA) ( 2– 6). In animals, most miRNAs are processed from a primary transcript (termed pri-miRNA) by two ribonuclease III (RNase III) enzymes, Drosha and Dicer. MicroRNAs (miRNAs) are ~22-nucleotide (nt) small RNAs that regulate dead-enylation, translation, and decay of their target mRNAs ( 1, 2).
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