Frequently Asked Questions
What is "miRNA=>ta-siRNA=>target" cleavage cascade?
Some microRNAs guide the cleavage of primary transcripts (precursors) of TAS gene (trans-acting siRNA gene, ta-siRNA). These microRNAs are called cleavage guider. After cleavage, the 3' (or 5')-side cleavage products are converted into double-stranded RNA (dsRNA) with the assistance of RDR6 and SGS3 and subsequently processed by DCL4 to produce a cluster of ta-siRNAs that are phased in 21-nt increment relative to the original cleavage site on both the strands. Eventually, the generated 21 nt ta-siRNAs further work as a component of RISC complex to guide AGO-dependent cleavage to their target transcript, i.e. prohibit the expression of their target gene at post-transcriptional level. The above mechanism bridges the miRNA with siRNA to form a cascade pathway.
Recent studies have reported that ta-siRNA also works as a cleavage guider to cut other TAS precursors just like as microRNA does, and then, generates more ta-siRNAs. Therefore, the whole cascade pathway could be represented as "miRNA => ta-siRNA => ta-siRNA => target gene" on some occassion.
Why do we focus on identification of ta-siRNA in the cascade?
According to the mechanism described above, ta-siRNA is a key component which links the whole cascade. Identifying ta-siRNA and its cleavage guider will profile the cascade pathway.
Why do we search potential ta-siRNAs from small RNA dataset?
MPSS and 454 are two kinds of massively sequencing platform used these days. The small RNAs are also being Increasingly sequenced via these two approaches. A typical MPSS library of Arabidopsis generally contain ~10K distinct 17bp small RNA sequences, which effectively profile the small RNAs of whole genome. Thus, a ta-siRNA could be efficiently discovered by searching these small RNAs, especially from certain special mutants.
How to find the phased small RNA clusters from small RNAs dataset.
ta-siRNA features a cluster of 21nt phased small RNAs which derive from the same TAS gene. Therefore, p-value test on the basis of hypergeometric distribution has proven to be an effective method in selecting the phased small RNA clusters from a larger number of small RNA sequences (Chen et al 2007).
Firstly, pssRNAMiner maps the input small RNAs on cDNA sequences and records their coordinates. Next, the algorithm slides on both strands of cDNA sequences to search each mapped small RNA and count the number of phased / non-phased position with small RNAs hits in a 231bp of downstream fragment from 5' start site of the small RNA. The following figure shows the region in which our algorithm counts phased/non-phased position with small RNA hits. The figure below is basically based on the figure 1 of Chen et al(2007 PMID: 17360645), however, a slight modification was made: We moved the phased point at the left end of negative strand to the right end, since each chop will generate a pair of opposite small RNAs.
Finally, the algorithm calculates the P-value of small RNA clusters based on the formula described by Chen et al(2007). Since phased small RNAs are often cut within 1-2 nt offset from phased positions, we introduce a variable, s , to reflect the 1-2 nt shift at phased positions in below fomula. This will also reduce the total number of non-phased position in the 231bp region.
How to identify valid cleavage site in a complementary region?
In biogenesis of a ta-siRNA, the cleavage site is a multiple of 21-nt away from the start site of generated ta-siRNAs (see figure below).
Meanwhile, the cleavage site should not be too far from the generated ta-siRNAs. That is to say, a microRNA is not necessarily always the cleavage guider of a TAS precursor, even if they have a complemetary region. Thus, in our algorithm, a complementary miRNA/ta-siRNA will be considered as a valid cleavage guider only if it fulfills the following two conditions:
(1) the user-predefined cleavage site in its complementary fragment must locate within a region calculated as following:
B1: start position of phased small RNAs cluster, B2: end position of phased small RNAs cluster, D: a user-specific maximum distance between cleavage site and start/end site of small RNAs cluster.
(2) the distance between the cleavage site and start site of the small RNA cluster must meet 21-nt phase property, i.e. a multiple of 21 nt.
* PMID represent ID number of PubMed, users could search the paper by PMID at http://www.ncbi.nlm.nih.gov/PubMed/
|© 2008 by The Samuel Roberts Noble Foundation, Inc.|