Middle Eocene to early Oligocene magnetostratigraphy of ODP Hole 711A (Leg 115), western equatorial Indian Ocean

<p>Ocean Drilling Program (ODP) Site 711, located in the western equatorial Indian Ocean near the Seychelles Archipelago on Madingley Rise, is an important site for studying middle Eocene to early Oligocene climatic evolution. This site is ideal for studying the impact of Neo-Tethyan gateway closure on Indian Ocean currents and circulation to further understand global climate changes through the greenhouse to icehouse transition. Middle Eocene-to-lower Oligocene strata recovered within Hole 711A (Cores 711A-14X to 21X) primarily consist of clay-bearing nannofossil oozes/chalks, with layers rich in radiolarians. Here, we report a high-resolution magnetostratigraphic record and a new integrated age model for the middle Eocene-to-lower Oligocene section of Hole 711A. Correlation of the polarity pattern to the geomagnetic polarity timescale provides a record from Chron C19r (middle Eocene) to C12r (early Oligocene). Our results extend the existing polarity record down into the middle Eocene and confirm published results from the lower Oligocene section of the hole. Overall, these new results from Hole 711A have important implications for identifying and dating global climate change events, and for reconstructing calcite compensation depth history at this site. </p

The siRNA Non-seed Region and Its Target Sequences Are Auxiliary Determinants of Off-Target Effects

<div><p>RNA interference (RNAi) is a powerful tool for post-transcriptional gene silencing. However, the siRNA guide strand may bind unintended off-target transcripts via partial sequence complementarity by a mechanism closely mirroring micro RNA (miRNA) silencing. To better understand these off-target effects, we investigated the correlation between sequence features within various subsections of siRNA guide strands, and its corresponding target sequences, with off-target activities. Our results confirm previous reports that strength of base-pairing in the siRNA seed region is the primary factor determining the efficiency of off-target silencing. However, the degree of downregulation of off-target transcripts with shared seed sequence is not necessarily similar, suggesting that there are additional auxiliary factors that influence the silencing potential. Here, we demonstrate that both the melting temperature (Tm) in a subsection of siRNA non-seed region, and the GC contents of its corresponding target sequences, are negatively correlated with the efficiency of off-target effect. Analysis of experimentally validated miRNA targets demonstrated a similar trend, indicating a putative conserved mechanistic feature of seed region-dependent targeting mechanism. These observations may prove useful as parameters for off-target prediction algorithms and improve siRNA ‘specificity’ design rules.</p></div

Analysis of off-target effects based on sequence similarity between an siRNA non-seed region and its corresponding target sequences.

<p><b>(A, B)</b> Cumulative distribution of off-target transcripts grouped by their non-seed base-pairing (Fig 4A; siVIM-270 off-target effects, 2AU (70), 1AU (52), None (27), 1GC (133), 2GC (151), Fig 4B; siVIM-805 off-target effects, 2AU (55), 1AU (52), None (20), 1GC (52), 2GC (46)). Off-target transcripts with more than 2AU or 2GC match were omitted due to their low number. <b>(C, D)</b> The average GC contents for non-seed region (positions 8–15) were calculated for each group of off-target transcripts. <b>(E, F)</b> The cumulative distribution of off-target transcripts of siVIM-270 with 1GC match (133 transcripts)(E) and 2GC matches (151 transcripts)(F), were sub-divided based on their GC contents at positions 8–15. ‘Low’ subgroups have GC content lower than the average while ‘High’ subgroups have GC content higher than the average.</p

Contribution of miRNA non-seed region to gene silencing.

<p>The list of putative targets perfectly matching the miRNA seed region (positions 2–8) was intersected with a list of experimentally validated miRNA targets [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004656#pcbi.1004656.ref023" target="_blank">23</a>]. Genes present on both lists were placed in the ‘Validated Targets’ group, while genes predicted to interact with miRNA but which have not been experimentally confirmed, were placed in ‘Remaining Genes’ group. The GC content in the positions 8–15 was calculated for both groups and compared. The difference was calculated by subtracting values of the ‘remaining’ group from the values in the ‘validated’ group.</p

Correlation between <i>T</i>_m values of sequence subsections within siRNA duplexes and the corresponding off-target silencing efficiency.

<p>The start of the subsection within a duplex is plotted on the Y axis (‘Start Position’) whereas the end of the subsection is plotted on the X axis (‘End Position’). The position numbering mirrors that used in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004656#pcbi.1004656.g001" target="_blank">Fig 1</a>. The analysis was performed separately for each siRNA concentration–<b>(A)</b> 0.05, <b>(B)</b> 0.5, <b>(C)</b> 5 and <b>(D)</b> 50 nM. The siRNA sequences used in the analysis, together with corresponding knockdown percentages, are listed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004656#pcbi.1004656.s001" target="_blank">S1 Table</a>.</p

Seed and non-seed region-dependent off-target effect analyses for siVIM-270 and siVIM-805.

<p><b>(A, B)</b> Expression profiles of off-target genes with 3’UTR sequences that perfectly match the corresponding siRNA seed region (i.e. off-targets) were compared to genes without such sequence. <b>(C, D)</b> The correlation between GC content in all the subsections within mRNA targets of siRNA non-seed region (8–21) and fold change of off-target effect was calculated in a similar manner to that shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004656#pcbi.1004656.g002" target="_blank">Fig 2</a>. The correlation coefficient was grouped using quantiles as boundary values and target position corresponds to the numbering shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004656#pcbi.1004656.g001" target="_blank">Fig 1</a>. GC contents in the non-seed regions at positions 8–15 showed the highest correlation with off-target effect (Fig 3C; siVIM-270, r = 0.22, p-value = 1.13E-12, Fig 3D; siVIM-805, r = 0.15, p-value = 1.57E-05). <b>(E, F)</b> Off-target transcripts for siVIM-270 and siVIM-805 were divided into four groups defined by the number of GC nucleotides in their non-seed regions (positions 8–15). Quantiles were used as the boundary values for classification; ‘Low’ (GC content < 3), ‘Medium’ (GC content = 3), ‘High’ (GC content = 4) and ‘Very High’ (GC content ≥ 5)(out of a total of 8). The number of off-target genes was 1065 for siVIM-270 (E) and 823 for siVIM-805 (F).</p