RNA repair enzymes catalyze rejoining of an RNA molecule after cleavage of phosphodiester linkages. RNA repair in budding yeast is catalyzed by two separate enzymes that process tRNA exons during their splicing and mRNA exons during activation of the unfolded protein response (UPR). The RNA ligase Trl1 joins 2',3'-cyclic phosphate and 5'-hydroxyl RNA fragments, creating a phosphodiester linkage with a 2'-phosphate at the junction. The 2'-phosphate is removed by the 2'-phosphotransferase Tpt1. We bypassed the essential functions of and in budding yeast by expressing "prespliced," intronless versions of the 10 normally intron-containing tRNAs, indicating this repair pathway does not have additional essential functions. Consistent with previous studies, expression of intronless tRNAs failed to rescue the growth of cells with deletions in components of the SEN complex, implying an additional essential role for the splicing endonuclease. TheΔ and Δ mutants accumulate tRNA and splicing intermediates indicative of RNA repair defects and are hypersensitive to drugs that inhibit translation. Failure to induce the unfolded protein response in Δ cells grown with tunicamycin is lethal owing to their inability to ligate after its cleavage by Ire1. In contrast, Δ mutants grow in the presence of tunicamycin despite reduced accumulation of spliced mRNA. We optimized a PCR-based method to detect RNA 2'-phosphate modifications and show they are present on ligated mRNA. These RNA repair mutants enable new studies of the role of RNA repair in cellular physiology.
In the unfolded protein response (UPR), stress in the endoplasmic reticulum (ER) activates a large transcriptional program to increase ER folding capacity. During the budding yeast UPR, Ire1 excises an intron from the HAC1 mRNA and the exon products of cleavage are ligated, and the translated protein induces hundreds of stress-response genes. Using cells with mutations in RNA repair and decay enzymes, we show that phosphorylation of two different HAC1 splicing intermediates is required for their degradation by the 5′→3′ exonuclease Xrn1 to enact opposing effects on the UPR. We also found that ligated but 2′-phosphorylated HAC1 mRNA is cleaved, yielding a decay intermediate with both 5′- and 2′-phosphates at its 5′-end that inhibit 5′→3′ decay and suggesting that Ire1 degrades incompletely processed HAC1. These decay events expand the scope of RNA-based regulation in the budding yeast UPR and have implications for the control of the metazoan UPR.
In the unfolded protein response (UPR), protein-folding stress in the endoplasmic reticulum (ER) activates a large transcriptional program to increase ER folding capacity. During the budding yeast UPR, the trans-ER-membrane kinase-endoribonuclease Ire1 excises an intron from the HAC1 mRNA and the exon cleavage products are ligated and translated to a transcription factor that induces hundreds of stress-response genes. HAC1 cleavage by Ire1 is thought to be the rate limiting step of its processing. Using cells with mutations in RNA repair and decay enzymes, we show that phosphorylation of two different HAC1 splicing intermediates by Trl1 RNA 5′-kinase is required for their degradation by the 5′→3′ exonuclease Xrn1 to enact opposing effects on the UPR. Kinase-mediated decay (KMD) of cleaved HAC1 3′-exon competes with its ligation to limit productive splicing and suppress the UPR, whereas KMD of the excised intron activates HAC1 translation, likely by relieving an inhibitory base-pairing interaction between the intron and 5′-untranslated region. We also found that ligated but 2′-phosphorylated HAC1 mRNA is endonucleolytically cleaved, yielding a KMD intermediate with both 5′-and 2′-phosphates at its 5′-end that inhibit 5′→3′ decay, and suggesting that Ire1 initiates the degradation of incompletely processed HAC1 s to proofread ligation or attenuate the UPR. These multiple decay events expand the scope of RNA-based regulation in the budding yeast UPR and may have implications for the control of the metazoan UPR by mRNA processing.
Liquid biopsies hold great promise in reducing the burden of the disease of cancer on patients. Early detection of cancer via the cell free DNA represents the broadest vision and requires highly specificity. Ongoing disease monitoring is another important application of liquid biopsy and requires high sensitivity. Assay development requires analytical validation, and assays must be monitored for ongoing performance. These assays demand reference materials at specific variant allele frequencies for the assessment of specificity and sensitivity. Previously, obtaining reference materials was a laborious process of finding or engineering mutant cell lines. Here, we describe the design, manufacture, and quality control of a synthetic reference standard: the Twist Pan-Cancer Reference Standards, developed using Twist’s proprietary DNA printing platform to include a wide selection of both common and rare cancer targets for analytical validation. The reference material closely mimics the size distribution and content of cell-free DNA, with a primary peak at 167 bp and a secondary peak at 334 bp. The background cfDNA is derived from a single donor and highly characterized through NGS. The reference standard includes over 400 variant sites across 84 genes, including literature-curated, clinically-relevant variant sites. Additional panel-wide variants were included to aid in troubleshooting capture panels. The variant DNA is synthetically printed at 167 bp ± 5 bp and tiles over the site with extensive overlap, providing diversity of DNA termini relative to the position of the site of variation. Variant sites were printed and quantified independently so they can be pooled uniformly. NGS quality controls are implemented at multiple steps to ensure quality. The final products are available in a variant allele frequency (VAF) dilution series. The dilution series is quality controlled using droplet digital PCR (ddPCR) to ensure precision of variant allele frequencies. The Twist Pan Cancer Reference Standards provide a valuable solution for NGS-based assay developers seeking reference materials for a wide array of cancer variants, advancing adoption of liquid biopsy tests toward routine clinical practice. Citation Format: Patrick D. Cherry. Twist pan-cancer synthetic reference materials for cell-free DNA (cfDNA) assay development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB110.
Introduction: DNA methylation at CpG nucleotide sites in eukaryotes is a key epigenetic mark that can help regulate gene expression. Specific changes in CpG methylation occur in many human cancers, making them a promising biomarker for early cancer detection. However, existing assays can be costly, lack specificity to regions of interest and often provide only semi-quantitative estimates of the methylation fraction. Here, we present a targeted methylome panel to decrease costs associated with next generation sequencing (NGS), methylation controls to calibrate quantitative assays and UMIs for accurate deduplication in low-diversity samples. Experimental Procedures: Genomic DNA (gDNA) was prepared for sequencing using the Twist Methylation Detection System consisting of enzymatic methylation conversion and hybrid capture using the Twist Human Methylome Panel. Twist’s synthetic CpG methylation level controls were spiked-in to gDNA and taken through the Twist Methylation Detection System to demonstrate their utility in calibrating methylation assays. Additionally, libraries were generated using cell free DNA (cfDNA) and either conventional or UMI-containing adapters to investigate the impact on quantitative detection and total unique coverage. Results: Using the Twist Human Methylome Panel at 150x raw coverage achieves uniform coverage with low off-bait Picard metrics. 6.59 million CpG sites were detected using a minimum depth of 10X. Target capture with the Human Methylome Panel allows for informative CpG calling of up to 82 samples on a single Illumina Novaseq S4 flowcell, compared to 3 samples per flowcell with a tradition whole-genome bisulfite sequencing (WGBS) assay. The Twist CpG methylation specific controls are constructed of 48 unique contrived sequences that contain a total of 8 different levels of methylation, ranging from 100% to 0%. Including these controls allows for quantitation of methylation levels in the experimental samples and qualification of the enzymatic conversion process. Conclusions: Our study leverages the Twist methylation detection portfolio to interrogate genome-wide methylation patterns for various applications. In combination, these tools can be used to design cost effective end-to-end assays. *For Research Use Only. Not for use in diagnostic procedures. Citation Format: Lydia Bonar, Kristin Butcher, Michael Bocek, Holly Corbitt, Bryan Hoglund, Cibelle Nassif, Patrick Cherry, Derek Murphy, Jean Challacombe, Esteban Toro. An end-to-end workflow for improved methylation detection. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6009.
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