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 RNAs, mainly including miRNA and phasiRNA (sRNA) sequences by the following standards:

  • A valid sequence can only be either FASTA or short tag format (see above figures);
  • At most 50M sRNA sequences can be analyzed once by pipeline and maximal submission size is 200MiB;
  • A valid small RNA sequence should be between 15 and 30 nt in length, and pipeline will ignore those invalid sequences;
  • Pipeline will further remove the small RNAs which are shorter than HSP size;
  • 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 5M target candidate sequences can be analyzed once by pipeline and maximal submission size is 1000MiB;
  • A valid target candidate sequence should be between 50 and 5,000,000 nucleotides 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 and their target transcript, we apply the scoring schema suggested by Axtell (2013) as preferred schema (V2, 2017update). Meanwhile, the oringinal scoring schema (V1, 2011update) in the first version of psRNATarget is also supported. 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):

The length of region in which the server will score complementarity between small RNA and target transcript. The preferred schema (V2) set this value to 19 since a few sRNA might be 19 nucleotides while the first version psRNATarget set it to 20. 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. In addition, the submitted small RNAs will be removed if they are shorted than HSP value.

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.

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.

Translation inhibition range:

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 sRNA/target pair with more target sites.