Nucleotide sequence of miRNA precursor contributes to cleavage site selection by Dicer

Abstract

The ribonuclease Dicer excises mature miRNAs from a diverse group of precursors (pre-miRNAs), most of which contain various secondary structure motifs in their hairpin stem. In this study, we analyzed Dicer cleavage in hairpin substrates deprived of such motifs. We searched for the factors other than the secondary structure, which may influence the length diversity and heterogeneity of miRNAs. We found that the nucleotide sequence at the Dicer cleavage site influences both of these miRNA characteristics. With regard to cleavage mechanism, we demonstrate that the Dicer RNase IIIA domain that cleaves within the 3′ arm of the pre-miRNA is more sensitive to the nucleotide sequence of its substrate than is the RNase IIIB domain. The RNase IIIA domain avoids releasing miRNAs with G nucleotide and prefers to generate miRNAs with a U nucleotide at the 5′ end. We also propose that the sequence restrictions at the Dicer cleavage site might be the factor that contributes to the generation of miRNA duplexes with 3′ overhangs of atypical lengths. This finding implies that the two RNase III domains forming the single processing center of Dicer may exhibit some degree of flexibility, which allows for the formation of these non-standard 3′ overhangs.

Somatic mutations in miRNA genes in lung cancer – potential functional consequences of non-coding sequence variants

“…It was shown before that the G>C substitution (SNP rs138166791) located at the same position significantly decreases the effectiveness of pre-miRNA processing and the level of mature miRNA (46). Additionally, as we have previously shown, such nucleotide substitutions may alter DROSHA cleavage specificity and may change the pool of generated isomiRs (85)(86)(87). Interestingly, although the minor allele of this SNP is very rare (<1% in the GnomAD, TOPMED, ExAC, and 1000G databases), we detected this allele as a hemizygous variant in 3 of ~60 lung cancer samples in our experimental analysis.…”

Comparing miRNA structure of mirtrons and non-mirtrons

“…These domains prefer to generate the U-ended miRNAs as the main fraction [63]. The G-ended miRNAs rarely occur and the G-avoiding generates the atypical 1nt and 3nt overhangs [64]. For the “homogeneous” cleavage (as it was defined by [63]) the overhang shortening appears as a 1nt context shift at the 3′ miRNA ends (compare figure 2A and figure 2С of the second-most frequent miRNA fraction in [63]).…”

“…Second, the pairs of long overhangs of the same sign are observed due to the incorrect prediction of terminal loop (Additional file 2) or to the presence of false miRNAs in the miRBase. And the last possibility is the guanine avoiding at the first position of 5′ end of 3′ miRNA [63, 64]. To exclude these three reasons, we further considered only the canonical overhangs and the overhangs with minimal 1nt deviations from them.…”

Newly identified miRNAs may contribute to aerenchyma formation in sugarcane roots

“…Usually, 21‐nt‐long mature sequences are a product of DCL1 or DCL4 (Voinnet, ). One of the nuclease domains, RNase III‐A, releases preferentially miRNAs with U as the 5’ terminal nucleotide (Starega‐Roslan, Galka‐Marciniak, & Krzyzosiak, ). Furthermore, miRNAs loading onto the Argonaute are also directed by the identity of the 5’ nucleotide in plants (Mi et al, ), humans (Frank, Sonenberg, & Nagar, ), and flies (Okamura, Liu, & Lai, ).…”