Prior to analysis, back-end pipeline will check submitted small RNA (mainly including miRNA and ta-siRNA, sic passim) sequences by the following standards:
Users are allowed to submit target candidate sequences of their interest in this section. A typical target transcript sequence can be a cDNA, EST, Unigene, mRNA or genomic segment, etc. The server will search possible target sites on these submitted target cadidates for (submitted or preloaded) small RNA sequences (mainly including miRNA and ta-siRNA, sic passim). Prior to analysis, back-end pipeline will check these submitted sequences by the following standards:
To score the complementarity between small RNA (mainly including miRNA and ta-siRNA, sic passim) and their target transcript, we apply the scoring schema of miRU by Zhang(Zhang 2005, PMID: 15980567). The maximum expectation is the threshold of the score. A small RNA/target site pair will be discarded if its score is greater than the threshold. The default cut-off threshold is 3.0. Users are advised to set more stringent cut-off threshold [0-2.0] for lower false positive prediction or more relaxed cut-off threshold [4.0-5.0] for higher prediction coverage.
Hspsize denotes the length of region in which the server will score complementarity between small RNA (including miRNA and ta-siRNA, sic passim) and target transcript. Zhang et al(2005) suggests that the hspsize value should be 20, which means server should score complementarity in the range of 1-20 nt of small RNA to the mRNA target site. However, some published miRNA mature sequences are shorter than 20 nt and original small RNA sequences generated Next-Generation Technology usually are shorter than 20 nt as well. Here, psRNATarget suggests users to decrease the value of hspsize if submitted sequences are shorter than 20 nt. Otherwise, these short sequences will be ignored by pipeline. Be aware that scoring algorithm will only penalize mismatches in this region(from No. 1 to No. hspsize nt) and subsequent mismatches (from hspsize nt to end) will be ignored.
The accessibility of mRNA target site to small RNA has been identified as one of important factors that are involved in target recognition because the secondary structure (stem etc.) around target site will prevent small RNA (including miRNA and ta-siRNA, sic passim) and mRNA target from contacting. The psRNATarget server employes RNAup to calculate target accessbility, which is represented by the energy required to open (unpair) secondary structure around target site (usually the complementary region with small RNA and up/downstream) on target mRNA(see figure below). The less energy means the more possibility that small RNA is able to contact (and cleave) target mRNA.
In above figure, represents the energy that is required to open secondary structure around target site. We use a software, namely RNAup, described by Muckstein et al (2005, pmid=16446276) to calculate this value, denoted as UPE.
Besides target site (complementary region with small RNA) itself, its two flanks on mRNA are also required to be opened in secondary structure for small RNA's (including miRNA and ta-siRNA, sic passim) binding and cleavage (see two red up-arrows in the following figure). The reason is that small RNA binds to target mRNA in the groove of RISC complex which need extra space on two sides of target site. Kertesz et al (2007)(PMID:17893677) suggested that 17 upstream and 13 downstream nucleotides of target site should be considered in target accessibility analysis.
In addition to cleave mRNA, plant miRNA also reportedly inhibits the translation of target genes. It often happens if any mismatch occurs in around center of complemetary region because the central region is essential for cleavage (Brodersen et al 2008, PMID: 18483398). This mechanism is different from translational inhibition of animal miRNA, although the latter also inhibits gene expression at the translational level.
Two-hits model (Axtell et al, 2005; PMID:17081978) suggests that a miRNA or ta-siRNA may have multiple target sites (i.e. complementary regions) on a specific target transcript, which will increase recognition actitivity of the miRNA/ta-siRNA to the mRNA target. The server will report the number of target sites for each small RNA/target pair. Users are advised to preferentially select a small RNA/target pair with more target sites.
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