20 research outputs found
Bioinformatics and biomolecular analyses indicate a role for APA in regulation of negative feedback.
<p>A) Comparison of APA-sites for HT miRNA host genes and NT miRNA host genes. B) After CPSF2 silencing HT miRNA host gene UTRs display a different poly(A)-site usage pattern compared to NT miRNA host gene UTRs and regular protein-coding genes’ UTRs. C) The motif discovered in upregulated APA regions after CPSF2 silencing resembles the two canonical polyadenylation sites. D) Distribution of canonical poly(A) signals across the 32UTR of HT miRNA host genes and E) NT miRNA host genes.</p
Model of intronic negative feedback regulation.
<p>After coexpression of miRNA and host gene, the miRNA directly regulates its host gene as well as CPSF2. After removal of CPSF2 the polyadenylation-complex is biased towards recognition of canonical sites. In the next transcription cycle, the canonical site that precedes the miRNA binding site is utilized. Hence, regulation of the host gene by its intronic miRNA is disabled.</p
Identification of APA genes preferentially targeted by HT miRNAs.
<p>Identification of APA genes preferentially targeted by HT miRNAs.</p
miR-579 targets its host, ZFR, and the APA associated gene CPSF2.
<p>A) Schematic diagram of the <i>ZFR</i> gene. B) Schematic diagram of the ZFR 32UTR including polyadenylation sites and the seed matching site for miR-579. C) U87 cells were co-transfected with reporter constructs containing wildtype ZFR-32UTR or ZFR-32UTR lacking the miR-579 binding site (mut 32UTR) along with pre-miR-579 or negative control (NC). Results are expressed as Rluc/Fluc ratio relative to NC (mean ± 95% CI; n = 6; *, p < 0.05). D) In U87 cells transiently transfected with scrambled control or pre-miR-579, ZFR and CPSF2 mRNA expression was analyzed by quantitative RT-PCR. Values are mean ± 95% CI; n = 5; *, p < 0.05. E) Western blot analysis of the same samples using specific antibodies as indicated (β-Actin served as loading control; one representative experiment of three is shown). F) In U87 cells, expression changes of the long (miRNA binding site containing; red) and short (without miRNA binding site; blue) alternatively polyadenylated UTRs after transfection with pre-miR-579 or with scrambled control was determined by quantitative RT-PCR. Values are shown as miR-579 transfection relative to scrambled control (n = 5; *, p < 0.05).</p
Estimated derived allele frequencies of retroCNVs segregating in three human subpopulations.
<p>Allele frequencies were calculated as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003242#s3" target="_blank">Materials and Methods</a>. RetroCNVs fixed in or absent from a given subpopulation are not shown.</p
HT miRNA host genes with significant 3´UTR changes after CPSF2-silencing.
<p>HT miRNA host genes with significant 3´UTR changes after CPSF2-silencing.</p
Detecting retroCNVs using sequence reads.
<p>a) RetroCNVs present in the reference genome are detected by searching for retrocopies in the reference that are absent from a sequenced individual, as revealed by paired-end reads spanning the location of the retroCNV and mapping too far apart from one another. b) RetroCNVs absent from the reference genome are detected by using paired-end reads to detect retroCNV insertion sites, and c) using reads that span exon-exon junctions but do not map to the reference genome.</p
RetroCNVs versus fixed retrocopies inserted in intronic versus intergenic sequence.
<p>RetroCNVs versus fixed retrocopies inserted in intronic versus intergenic sequence.</p
RetroCNVs versus fixed retrogenes moving from an autosome to an autosome (A→A) from the X chromosome to the X (X→X), from the X to the autosomes (X→A), or vice-versa (A→X).
*<p>Data from Emerson et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003242#pgen.1003242-Emerson2" target="_blank">[29]</a>.</p