To support the psRNATarget, please cite: Xinbin Dai and Patrick X. Zhao, psRNATarget: A Plant Small RNA Target Analysis Server, Nucleic Acids Research, 2011, W155-9. doi: 10.1093/nar/gkr319.
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---- A Plant Small RNA Target Analysis Server       
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Data formats allowed by psRNATarget:

FASTA format:
Short Tags: for small RNA sequences, one sequence per line
Pure Sequence: a single sequence without FASTA head (may occupy multi-lines)

User-submitted small RNA sequence(s):

Prior to analysis, back-end pipeline will check submitted small RNA (mainly including miRNA and ta-siRNA, sic passim) sequences by the following standards:

  • A valid sequence can only be either FASTA or short tag format (see above figures);
  • At most 5000000 sequences can be analyzed once by pipeline and maximal submission size is 200M;
  • A valid small RNA sequence should be between 15 and 30 nt in length, and pipeline will ignore those invalid sequences;
  • Only 'ATCGUN' are valid sequence letters, a sequence containing other letters will be ignored;
For FASTA format, we only suggest alphabet, digit and minor/underscore characters in sequence ID. In addition, please avoid long sequences ID, such as, a ID longer than 50 letters, because long ID may mess up web display.

User-submitted target candidate sequence(s):

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:

  • A valid sequence can only be FASTA format or single sequence without FASTA header(Pure Sequence, see above figures);
  • At most 5000000 target candidate sequences can be analyzed once by pipeline and maximal submission size is 1000M;
  • A valid target candidate sequence should be between 50 and 5000000 nt in length, and pipeline will ignore those invalid sequences;
  • Only 'ATCGUN' are valid sequence letters, other characters will be deleted or changed to 'N'.
For FASTA format, we only suggest alphabet, digit and minor/underscore characters in sequence ID. In addition, please avoid long sequences ID, such as, a ID longer than 50 letters, because long ID may interrupt web display.

Maximum expectation:

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.

Length for complementarity scoring (hspsize):

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.

Number of top target genes for each small RNA (top):

The number of top (the best) target gene candidates that will be listed for each submitted small RNA.

Note: The value had been set as 25. Per users requests, this parameter was made accessible to end users with the default value 200 in June 2014.

Target accessibility - maximum energy to unpair the target site (UPE):

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.

Flanking length around target site for target accessibility analysis:

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.

Range of central mismatch leading to translational inhibition:

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.

The users are allowed to set coordinates of central region in which any mismatch will be reported as the trigger of translational inhibition.

multiplicity of target site:

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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