SIREs (searching for IREs) is an improved bioinformatic program that detect iron-responsive elements-like motifs based on present and previous iron-responsive elements binding studies [1,2,3 and Sanchez et al. manuscript in preparation]. SIREs program is a Perl-based program that screen for a 19 or 20 nucleotide sequence corresponding to the core sequence of an iron-responsive elements (nucleotides n07 to n25 squared in the below figure).
Previous SELEX (systematic evolution of ligands by exponential enrichment) experiments have reported that the 6 nucleotide apical loop of an iron-responsive element can differ from the canonical CAG(U/A)GN sequence [1,2,3]. Our programme takes into consideration this variability and allows a total of 18 motifs (2 canonical and 16 SELEX motifs) proven to bind Iron Regulatory Protein 1 and/or Iron Regulatory Protein 2 in vitro with a relative binding efficiency bigger than 20%. The new 16 SELEX motifs differ from the canonical sequence in the sequence of the 6 nucleotide apical loop of an IRE (positions n14 to n19) or at the C bulge (n8 is a G in motif 18) (See table below). Some of these motifs have been reported to be specific for the binding of IRP1 or IRP2 (see specificity column in the table).
Because the IREs of Dmt1 and Hif2aplha have one bulge nucleotide at the right side of the upper stem (position n21b, U, see red arrow in figure below), we design the SIREs program in order to be able to detect similar type of IREs allowing one single bulge nucleotide at positions n20b, n21b, n22b or n23b. In addition, SIREs program allow the detection of IRE-like motif with a mistmach in the upper stem (positions n13-n20, n12-n21, n11-n22, n10-n23, n09-n24 or n07-n25), similarly as the one present in the Gox mRNA (see red arrow in figure below) [4]. The detection of one bulge nucleotide at the right side of the upper stem or a mismatch in the upper stem or at position n07-n25 is mutually exclusive.
Predicted IRE motifs are reported as SIREs Predictions that include the 19 or 20 nucleotide sequence corresponding to positions n07 to n25. Additional 6 nucleotides from the lower stem are also reported. The RNA Folding predicted by the RNAfold Program of the Vienna Package is also reported for the predicted IRE.
The following information is provided for each predicted IRE:
- Motif type: it refers to nucleotide sequence present at positions n8 (normally the C8 bulge) and at positions n14 to n19. See table above.
- Mismatch: it refers to the possible mismatch found at positions positions n13-n20, n12-n21, n11-n22, n10-n23, n09-n24 or n07-n25. Base pair mismatch are reported.
- Upper stem Bulge: it refers to the bulge nucleotide at the right side of the upper stem (position n20b, n21b, n22b or n23b.
- Apical loop: it refers to the six nucleotides of the apical loop, position n14, n15, n16, n17 , n18 and n19.
- N25: it refers to the nucleotide at position n25. The presence of a G nucleotide at this position should be taken with caution since it may pair with the C8 nucleotide and hence impair the formation of a proper IRE.
- Number of GU Pairs: it denotes the numbers of wobble base pair G.U or U.G in the upper stem or at position n07-n25. This number should be 0, 1 or 2 since we experimentally have shown that 3 or more wobble base pairs impair the formation of a proper IRE (Sanchez et al., manuscript in preparation).
- Free Energy: it denotes the minimum free energy of the predicted IRE motif calculated by RNAfold Program . Minimum free energy of known IREs range between –9.8 units to –1.4 Kcal/mol.
- Level of stringency (High, Medium or Low): According to our experience we have set 3 levels of stringency for the prediction of IREs. Report features have been scored and depending on the sum value of the scores IREs are classified in the 3 categories:
- Scores from 8 to 6: overall quality: High
- Scores from 5.9 to 4: overall quality: Medium
- Scores from 3.9 to 0: overall quality: Low
Sensitivity, specificity and precision of the SIREs program for the three different stringent levels have been calculated using a data set of 32 novel IRP-target genes recently identified enriched on IRE-containing mRNA (Sanchez et al., manuscript in preparation) and 150 random sequences that are not expected to harbor IRE elements. Values are reported in the below table.
| High | Medium | Low | |
|---|---|---|---|
| Sensitivity | 0.28 | 0.44 | 0.56 |
| Specificity | 0.99 | 0.95 | 0.91 |
| Precision | 0.90 | 0.67 | 0.58 |
| Recall | 0.87 | 0.88 | 0.91 |
In summary, our SIREs program represents an improved software of the currently available programs that can predict IRE sequences [7,8] since it is able to detect IRE-like sequences with a combination of 18 motifs in the 6 nucleotide apical loop, one single mismatch in the upper stem or at position n07-n25 or one single bulge nucleotide at the right side of the upper stem.