Predicting off-target effects of antisense oligomers targeting bacterial mRNAs with the MASON webserver
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ABSTRACT:
PROVIDER: PRJNA820711 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR18503000.fastq.gz | Fastqsanger.gz | |||
| SRR18503001.fastq.gz | Fastqsanger.gz | |||
| SRR18503002.fastq.gz | Fastqsanger.gz | |||
| SRR18503003.fastq.gz | Fastqsanger.gz | |||
| SRR18502976.fastq.gz | Fastqsanger.gz |
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Project description:Antisense oligomers (ASOs) such as peptide nucleic acids (PNAs), designed to inhibit the translation of essential bacterial genes, have emerged as attractive sequence- and species-specific programmable RNA antibiotics. Yet, potential drawbacks include unwanted side effects caused by their binding to transcripts other than the intended target. To facilitate the design of PNAs with minimal off-target effects, we developed MASON (Make AntiSense Oligomers Now), a webserver for the design of PNAs that target bacterial mRNAs. MASON generates PNA sequences complementary to the translational start site of a bacterial gene of interest and reports critical sequence attributes and potential off-target sites. We based MASON’s off-target predictions on experiments in which we treated Salmonella enterica serovar Typhimurium with a series of 10mer PNAs derived from a PNA targeting the essential gene acpP but carrying two serial mismatches. Growth inhibition and RNA-sequencing (RNA-seq) data revealed that PNAs with terminal mismatches are still able to target acpP, suggesting wider off-target effects than anticipated. Comparison of these results to an RNA-seq dataset from uropathogenic Escherichia coli (UPEC) treated with eleven different PNAs confirmed our findings are not unique to Salmonella. We believe that MASON’s off-target assessment will improve the design of specific PNAs and other ASOs.
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Project description:Optimized design of antisense oligomers for targeted rRNA depletion
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Project description:To identify the circNEIL3 interacting protein, we performed RNA pull-down assays after transfecting into cells with biotin-labeled sense (S) or antisense (AS) DNA oligomers crossing the junction site of circNEIL3, and the pull-down proteins were subjected to SDS-PAGE and Coomassie blue staining. ~45 kDa Band pulled down by the AS probe was specifically excised , digested and used for mass spectrometry analysis.
2023-10-24 | PXD039394 | Pride
Project description:Antisense transcription is a pervasive phenomenon, but its source and functional significance is largely unknown. We took an expression-based approach to explore microRNA (miRNA)-related antisense transcription by computational analyses of published whole-genome tiling microarray transcriptome and deep sequencing small RNA (smRNA) data. Statistical support for greater abundance of antisense transcription signatures and smRNAs was observed for miRNA targets than for paralogous genes with no miRNA cleavage site. Antisense smRNAs were also found associated with MIRNA genes. This suggests that miRNA-associated "transitivity" (production of small interfering RNAs through antisense transcription) is more common than previously reported. High-resolution (3 nt) custom tiling microarray transcriptome analysis was performed with probes 400 bp 5' upstream and 3' downstream of the miRNA cleavage sites (direction relative to the mRNA) for 22 select miRNA target genes. We hybridized RNAs labeled from the smRNA pathway mutants, including hen1-1, dcl1-7, hyl1-2, rdr6-15, and sgs3-14. Results showed that antisense transcripts associated with miRNA targets were mainly elevated in hen1-1 and sgs3-14 to a lesser extent, and somewhat reduced in dcl11-7, hyl11-2, or rdr6-15 mutants. This was corroborated by semi-quantitative reverse transcription PCR; however, a direct correlation of antisense transcript abundance in MIR164 gene knockouts was not observed. Our overall analysis reveals a more widespread role for miRNA-associated transitivity with implications for functions of antisense transcription in gene regulation. HEN1 and SGS3 may be links for miRNA target entry into different RNA processing pathways.
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Project description:MicroRNA targets are often recognized through pairing between the miRNA seed region and complementary sites within target mRNAs, but not all of these canonical sites are equally effective, and both computational and in vivo UV-crosslinking approaches suggest that many mRNAs are targeted through non-canonical interactions. Here, we show that recently reported non-canonical sites do not mediate repression despite binding the miRNA, which indicates that the vast majority of functional sites are canonical. Accordingly, we developed an improved quantitative model of canonical targeting, using a compendium of experimental datasets that we pre-processed to minimize confounding biases. This model, which considers site type and another 14 features to predict the most effectively targeted mRNAs, performed significantly better than existing models and was as informative as the best high-throughput in vivo crosslinking approaches. It drives the latest version of TargetScan (v7.0; targetscan.org), thereby providing a valuable resource for placing miRNAs into gene-regulatory networks.
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Project description:We investigated whether pharamacological inhibition of Smyd3 by antisense oligonucleotides (ASOs) can influence diethylnitrosamine (DEN)-induced liver cancer development. Our phenotypic analyses, among others, included gene expression profiling.
2020-06-24 | GSE153077 | GEO
Project description:RNA sequencing (RNA-seq) has become a standard method for quantifying gene expression transcriptome-wide. Due to the extremely high proportion of ribosomal RNA (rRNA) in total RNA, sequencing libraries usually incorporate messenger RNA (mRNA) enrichment. Although polyadenylate (poly(A)) tail selection is widely used, many applications require alternate approaches such as rRNA depletion. Recently, selective rRNA digestion, using RNaseH and antisense DNA oligomers that tile the length of target RNAs, has emerged as an easy, cost-effective alternative to commercial rRNA depletion kits. Here, we present a streamlined RNaseH-mediated rRNA depletion method that uses shorter antisense oligos that only sparsely tile the target RNA, in a digestion reaction of only 5 minutes. We wrote a Web tool, Oligo-ASST, that simplifies oligo design to favor target regions with optimal thermodynamic properties, and additionally allows users to design common oligo pools that can simultaneously target divergent RNAs in their regions of higher sequence similarity. We demonstrate the efficacy of these oligos by building rRNA-depleted sequencing libraries for Xenopus laevis as well as zebrafish, which expresses two distinct versions of the 28S, 18S, 5.8S, and 5S rRNAs during embryogenesis. These libraries efficiently deplete rRNA to <5% of total reads, on par with poly(A) selection, and also reveal expression of many non-adenylated RNA species. Oligo-ASST is freely available at https://mtleelab.pitt.edu/oligo to design antisense oligos for any taxon or to target any abundant RNA for depletion.
2020-06-28 | GSE152902 | GEO
Project description:Regulation of mRNA stability by RNA-protein interactions contributes significantly to quantitative aspects of gene expression. We have identified potential mRNA targets of the AU-rich element binding protein AUF1. Myc-tagged AUF1 p42 was induced in mouse NIH-3T3 cells and RNA-protein complexes isolated using anti-myc tag antibody beads. Bound mRNAs were analyzed with Affymetrix microarrays. We have identified 508 potential target mRNAs that were at least 3-fold enriched compared to control cells without myc-AUF1. 22.3% of the enriched mRNAs had an AU-rich cluster in the ARED Organism database, against 16.3% of non-enriched control mRNAs. The enrichment towards AU-rich elements was also visible by AREScore with an average value of 5.2 in the enriched mRNAs versus 4.2 in the control group. Yet, many mRNAs were enriched without a high ARE score suggesting that AUF1 has a broader binding spectrum than standard AUUUA repeats. AUF1 did not preferentially bind to unstable mRNAs. Still, some enriched mRNAs were highly unstable, as those of TNFSF11 (known as RANKL), KLF10, HES1, CCNT2, SMAD6, and BCL6. We have mapped some of the instability determinants. HES1 mRNA appeared to have a coding region determinant. Detailed analysis of the RANKL and BCL6 3’UTR revealed for both that full instability required two elements, which are conserved in evolution. In RANKL mRNA both elements are AU-rich and separated by 30 bases, while in BCL6 mRNA one is AU-rich and 60 bases from a non AU-rich element that potentially forms a stem-loop structure.
2015-09-30 | GSE64761 | GEO
Project description:RNase H1-dependent antisense oligonucleotides (ASOs) can degrade complementary RNAs in both the nucleus and the cytoplasm. Since cytoplasmic mRNAs are actively engaged in translation, ASO activity may thus be affected by translating ribosomes that scan the mRNAs. Here we show that mRNAs associated with ribosomes can be cleaved using ASOs and that translation can alter ASO activity. Translation inhibition tends to increase ASO activity when targeting the coding regions of efficiently translated mRNAs, but not nuclear non-coding RNAs or less efficiently translated mRNAs. Increasing the level of RNase H1 protein eliminated the enhancing effects of translation inhibition on ASO activity, suggesting that RNase H1 recruitment to ASO/mRNA heteroduplexes is a rate limiting step and that translating ribosomes can inhibit RNase H1 recruitment. Consistently, ASO activity was not increased by translation inhibition when targeting the 3' UTRs, independent of the translation efficiency of the mRNAs. Contrarily, the activity of 3' UTR-targeting ASOs tended to be reduced upon translation inhibition, likely due to decreased accessibility. These results indicate that ASO activity can be affected by the translation process, and the findings also provide important information toward helping better ASO drug design.
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Project description:Antisense peptide nucleic acids (PNAs) that target mRNAs of essential bacterial genes exhibit specific bactericidal effects in several microbial species, but our mechanistic understanding of PNA activity and their target gene spectrum is limited. Here, we present a systematic analysis of PNAs targeting eleven essential genes with varying expression levels in uropathogenic Escherichia coli (UPEC). We demonstrate that UPEC is susceptible to killing by peptide-conjugated PNAs, especially when targeting the widely-used essential gene acpP. Our evaluation yields three additional promising target mRNAs for effective growth inhibition, i.e., dnaB, ftsZ, and rpsH. The analysis also shows that transcript abundance does not predict target vulnerability and that PNA-mediated growth inhibition is not universally associated with target mRNA depletion. Global transcriptomic analyses further reveal PNA sequence-dependent but also -independent responses, including the induction of envelope stress response pathways. Importantly, we show that the growth inhibitory capacity of 9mer PNAs is generally as effective as their 10mer counterparts. Overall, our systematic comparison of a range of PNAs targeting mRNAs of different essential genes in UPEC suggests important features for PNA design, reveals a general bacterial response to PNA conjugates and establishes the feasibility of using PNA antibacterials to combat UPEC.
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