Mature microRNA-binding protein QKI promotes microRNA-mediated gene silencing
February 2024
Authors:
Kyung-Won Min, Myung Hyun Jo, Minseok Song, Ji Won Lee, Min Ji Shim, Kyungmin Kim, Hyun Bong Park,Shinwon Ha, Hyejin Mun, Ahsan Polash, Markus Hafner, Jung-Hyun Cho, Dongsan Kim, Ji-Hoon Jeong, Seungbeom Ko, Sungchul Hohng, Sung-Ung Kang & Je-Hyun Yoon
Abstract:
“Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.“
Sage Science Products:
PippinHT was used to isolate micro RNA libraries.
Methods Excerpt:
“Library preparation was performed using the TruSeq Small RNA library preparation kit (Illumina, RS-200) following the manufacturer’s instructions. Briefly, 1 μg of input RNA was loaded into Urea-TBE gels for purification and the resulting RNAs were applied for the following procedures. User-supplied reagents including T4 RNA ligase2 Deletion Mutant (Lucigen, LR2D1132K) and Maxima First Stand cDNA synthesis kit (Thermo Fisher Scientific, K1641) were purchased separately. Libraries were amplified using 11 cycles of PCR for the manufacture’s index or modified index primer set to increase diversity. Libraries were prepared according to the manufacturer’s protocol with a modification in the size selection step, which, instead of agarose gel purification, PippinHT Prep instrument (Sage Science, HTP0001) and 3% agarose dye-free cassette with internal standards (Sage Science, HTG3010) was used under the following conditions: base pair start = 120 bp, base pair end = 160 bp, range = broad, target peak size = 145 bp. Eluted Libraries from PippinHT system were subsequently analysed on Tape-station 4150 (Agilent Technologies, G2992AA) following the manufacturer’s instructions using a High Sensitivity DNA Screen tape (Agilent Technologies, 5067–5584). Each library was barcoded with unique sequence of reverse primers during the PCR step, which contained Illumina compatible indices and modified indexes (see GEO database). Before pooling libraries for the next-generation sequencing, concentration of each library was measured in a high sensitivity Tape-station followed by smear analysis. Illumina NextSeq 550 or Miniseq with single end (50 nt;R1) read method was apply for the library sequencing.”
Author Affiliations:
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC
Department of Biology,Gangneung-Wonju National University, Gangneung, Republic of Korea
Department of Physics & Astronomy, Seoul National University, Seoul,Republic of Korea
Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD
Department of Oncology Science, University of Oklahoma, Oklahoma City, OK
Laboratory of Muscle Stem Cells and GeneRegulation, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD
Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul, Korea
RNA Biology
DOI: 10.1080/15476286.2024.2314846
In vitro size-selection of short circulating tumor DNA fragments from late-stage lung cancer patients enhance the detection of mutations and aneuploidies
January 2024
Authors:
Christoffer Trier Maansson, Louise Skov Thomsen, Laura Stokkebro, Julie Gabe Dissing, Maiken Parm Ulhoi, Anders Lade Nielsen, Peter Meldgaard, Boe Sandahl Sorensen
Abstract:
“Introduction
Recent studies have demonstrated differences between the fragment length profiles of cell-free DNA (cfDNA) from cancer patients and healthy individuals. This has led to the development of in vitro size-selection procedures which can isolate the short fragments that are enriched with mutated circulating tumor DNA (ctDNA). This has yet to be investigated in a large cohort of lung cancer patients.
Materials and methods
We used plasma samples from 35 stage III and IV lung cancer patients and performed targeted next-generation sequencing (NGS) and variant calling from cfDNA with and without size-selection of short fragments. We identified clonal hematopoiesis (CH) and germline mutations using targeted NGS on paired buffy coat (BC) samples. In addition, we performed a genome-wide copy-number alteration analysis on the cfDNA samples with and without size-selection.
Results
ctDNA containing tumor mutations had a different fragment length profile compared to cfDNA fragments with CH or germline mutations. In vitro size-selection resulted in a median 1.36-fold (interquartile range (IQR): 0.63 to 2.48) mutational allele fraction (MAF) enrichment of tumor mutations whereas CH/germline mutations had a median 0.95-fold (IQR: 0.62 to 1.05) MAF enrichment. Key oncogenic drivers, including KRAS and EGFR were more likely to have a MAF increase with size-selection. Size-selection also increased the number plasma aneuploidy positive samples from 8 of 35 to 20 of 35.
Conclusion
This study expands the knowledge regarding ctDNA fragmentation in lung cancer patients and we demonstrate that in vitro size-selection can increase MAF of tumor mutations and plasma aneuploidy calls. Size-selection could lead to increased sensitivity of ctDNA detection, which is crucial for clinical implementation of liquid biopsies. This study is the largest of its kind studying cfDNA samples from 35 lung cancer patients containing 109 mutations in total.. “
Sage Science Products:
PippinHT was used to size select cfDNA samples.
Methods Excerpt:
“In vitro size-selection of cfDNA was performed using PippinHT (Sage Science, Beverly, MA, USA) according to the manufacturer’s instructions. For each sample, 20 μl of purified cfDNA was added to a 3 % agarose gel cassette. The range mode was used to collect fragments with 95–230 bp with a pause at 152 bp. This resulted in a collection of two 30 μl cfDNA fractions and the fraction containing the short fragments (95–152 bp) was subjected to quality control and CAPP-seq..”
Author Affiliations:
Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark
Department of Clinical Medicine, Aarhus University, Aarhus, Denmark
Department of Biomedicine, Aarhus University, Aarhus, Denmark
Department of Oncology, Aarhus University Hospital, Aarhus, Denmark
The Journal of Liquid Biopsy
DOI: 10.1016/j.jlb.2024.100141
The haplotype-resolved T2T carnation (Dianthus caryophyllus) genome reveal the correlation between genome architecture and gene expression
November 2023
Authors:
Lan Lan, Luhong Leng, Weichao, Yonglin Ren, Wayne Reeve, Xiaopeng Fu, Zhiqiang Wu, Xiaoni Zhang
Abstract:
“Carnation (Dianthus caryophyllus) is one of the most valuable commercial 45 flowers, due to its richness of colour and form, and its excellent storage and vase life. 46 The diverse demands of the market require faster breeding in carnations. A full 47 understanding of carnations is therefore required to guide the direction of breeding. 48 Hence, we assembled the haplotype-resolved gap-free carnation genome of a variety 49 ‘Baltico’ which is the most common white standard variety worldwide. Based on the 50 high-depth HiFi, ultra-long nanopore and Hi-C sequencing data, we assembled the 51 telomere-to-telomere (T2T) genomes to be 564,479,117 and 568,266,215 bp, for the 52 two haplotypes Hap1 and Hap2, respectively. This T2T genome exhibited great 53 improvement in genome assembly and annotation results compared with the former 54 version. The improvements were seen when different approaches to evaluation were 55 used. Our T2T genome first informs the analysis of the telomere and centromere 56 region, enabling us to speculate about the specific centromere characteristics that 57 cannot be identified by high order repeats in carnations. We analyzed the allele-58 specific expression in three tissues and the relationship between the genome 59 architecture and gene expression in the haplotypes. This demonstrated that the length 60 of the genes, CDS, introns, the exon numbers and the transposable elements insertions 61 correlate with gene expression ratios and levels. The insertions of transposable 62 elements repress expression in gene regulatory networks in carnation. This gap-free 63 finished T2T carnation genome provides a valuable resource to illustrate the genome 64 characteristics and functional genomics analysis for further studies and molecular 65 breeding. “
Sage Science Products:
The SageHLS instrument was used to size select Ultra High Molecular Weight DNA for Oxford Nanopore Promethion sequencing (Genome Center of Grandomics, Wuhan China)
Author Affiliations:
Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Chinese Academy of Agricultural Sciences, Shenzhen, China
College of Science, Health, Engineering and Education, Murdoch University, Western Australia
Kunpeng Institute of Modern Agriculture at Foshan, Shenzhen Branch, Chinese Academy of Agricultural Sciences, Shenzhen, China
Key Laboratory of Horticultural Plant Biology, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, China
Preprint – Horticulture Research
DOI: 10.1093/hr/uhad244
The draft genome sequence of the Japanese rhinoceros beetle Trypoxylus dichotomus septentrionalis towards an understanding of horn formation
May 2023
Authors:
Shinichi Morita, Tomoko F. Shibata, Tomoaki Nishiyama, Yuuki Kobayashi, Katsushi Yamaguchi, Kouhei Toga, Takahiro Ohde, Hiroki Gotoh, Takaaki Kojima, Jesse N. Weber, Marco Salvemini, Takahiro Bino, Mutsuki Mase, Moe Nakata, Tomoko Mori, Shogo Mori, Richard Cornette, Kazuki Sakura, Laura C. Lavine, Douglas J. Emlen, Teruyuki Niimi and Shuji Shigenobu
Abstract:
“The Japanese rhinoceros beetle Trypoxylus dichotomus is a giant beetle with distinctive exaggerated horns present on the head and prothoracic regions of the male. T. dichotomus has been used as a research model in various fields such as evolutionary developmental biology, ecology, ethology, biomimetics, and drug discovery. In this study, de novo assembly of 615 Mb, representing 80% of the genome estimated by flow cytometry, was obtained using the 10 × Chromium platform. The scaffold N50 length of the genome assembly was 8.02 Mb, with repetitive elements predicted to comprise 49.5% of the assembly. In total, 23,987 protein-coding genes were predicted in the genome. In addition, de novo assembly of the mitochondrial genome yielded a contig of 20,217 bp. We also analyzed the transcriptome by generating 16 RNA-seq libraries from a variety of tissues of both sexes and developmental stages, which allowed us to identify 13 co-expressed gene modules. We focused on the genes related to horn formation and obtained new insights into the evolution of the gene repertoire and sexual dimorphism as exemplified by the sex-specific splicing pattern of the doublesex gene. This genomic information will be an excellent resource for further functional and evolutionary analyses, including the evolutionary origin and genetic regulation of beetle horns and the molecular mechanisms underlying sexual dimorphism.”
Sage Science Products:
The SageHLS instrument was used to size select DNA between 50-80 kb for 10X Genomics Chromium linked read analysis.
Author Affiliations:
Division of Evolutionary Developmental Biology, National Institute for Basic Biology, Okazaki, Japan
Department of Basic Biology, School of Life Science, The Graduate University for Advanced Studies, SOKENDAI, Okazaki, Japan
Division of Evolutionary Biology, National Institute for Basic Biology, Okazaki, Japan
Division of Integrated Omics Research, Research Center for Experimental Modeling of Human Disease, Kanazawa University, Kanazawa, Japan
Laboratory of Evolutionary Genomics, National Institute for Basic Biology, Okazaki, Japan
Trans-Omics Facility, National Institute for Basic Biology, Okazaki, Japan
Laboratory of Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
URA Division, Office of Research and Academia-Government-Community Collaboration, Hiroshima University, Hiroshima, Japan
Department of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
Department of Biological Science, Faculty of Science, Shizuoka University, Shizuoka, Japan
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
Department of Agrobiological Resources, Faculty of Agriculture, Meijo University, Nagoya, Japan
Department of Integrative Biology, University of Wisconsin-Madison, Madison, WI, USA
Department of Biology, University of Naples Federico II, Naples, Italy
Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Japan
Department of Entomology, Washington State University, Pullman, WA, USA
Division of Biological Sciences, The University of Montana, Missoula, MT, USA
Citation
Nature Scientific Reports
DOI:10.1038/s41598-023-35246-w
Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library Method
March 2023
Authors:
Veronika Mikhaylova, Madison Rzepka, Tetsuya Kawamura1, Yu Xia, Peter L. Chang, Shiguo Zhou, Long Pham, Naisarg Modi, Likun Yao, Adrian Perez-Agustin, Sara Pagans, T. Christian Boles , Ming Lei, Yong Wang, Ivan Garcia-Bassets, and Zhoutao Chen
Abstract:
“In the human genome, heterozygous sites are genomic positions with different alleles inherited from each parent. On average, there is a heterozygous site every 1-2 kilobases (kb). Resolving whether two alleles neighboring heterozygous positions are physically linked—that is, phased—is possible with a short-read sequencer if the sequencing library captures long-range information. TELL-Seq is a library preparation method based on millions of barcoded micro-sized beads that enables instrument-free phasing of a whole human genome in a single PCR tube. TELL-Seq incorporates a unique molecular identifier (barcode) to the short reads generated from the same high-molecular-weight (HMW) DNA fragment (known as ‘linkedreads’). However, genome-scale TELL-Seq is not cost-effective for applications focusing on a single locus or a few loci. Here, we present an optimized TELL-Seq protocol that enables the cost-effective phasing of enriched loci (targets) of varying sizes, purity levels, and heterozygosity. Targeted TELL-Seq maximizes linked-read efficiency and library yield while minimizing input requirements, fragment collisions on microbeads, and sequencing burden. To validate the targeted protocol, we phased seven 180-200 kb loci enriched by CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis, four 20 kb loci enriched by CRISPR/Cas9-mediated protection from exonuclease digestion, and six 2-13 kb loci amplified by PCR. The selected targets have clinical and research relevance (BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, and PKI3CA). These analyses reveal that targeted TELL-Seq provides a reliable way of phasing allelic variants within targets (2-200 kb in length) with the low cost and high accuracy of short-read sequencing. Lynch syndrome (LS), caused by heterozygous pathogenic variants affecting one of the mismatch repair (MMR) genes (MSH2, MLH1, MSH6, PMS2), confers moderate to high risks for colorectal, endometrial, and other cancers. We describe a four-generation, 13-branched pedigree in which multiple LS branches carry the MSH2 pathogenic variant c.2006G>T (p.Gly669Val), one branch has this and an additional novel MSH6 variant c.3936_4001+8dup (intronic), and other non-LS branches carry variants within other cancer-relevant genes (NBN, MC1R, PTPRJ). Both MSH2 c.2006G>T and MSH6 c.3936_4001+8dup caused aberrant RNA splicing in carriers, including out-of-frame exon-skipping, providing functional evidence of their pathogenicity. MSH2 and MSH6 are co-located on Chr2p21, but the two variants segregated independently (mapped in trans) within the digenic branch, with carriers of either or both variants. Thus, MSH2 c.2006G>T and MSH6 c.3936_4001+8dup independently confer LS with differing cancer risks among family members in the same branch. Carriers of both variants have near 100% risk of transmitting either one to offspring. Nevertheless, a female carrier of both variants did not transmit either to one son, due to a germline recombination within the intervening region. Genetic diagnosis, risk stratification, and counseling for cancer and inheritance were highly individualized in this family. The finding of multiple cancer-associated variants in this pedigree illustrates a need to consider offering multicancer gene panel testing, as opposed to targeted cascade testing, as additional cancer variants may be uncovered in relatives.”
Sage Science Products:
The HLS-CATCH process (SageHLS system) was used to purify the larger (180-200kb) targets.
Author Affiliations:
Universal Sequencing Technology Corp., Carlsbad, CA
Sage Science Inc., Beverly, MA
Department of Medicine, University of California, San Diego, La Jolla, CA
Department of Medical Sciences, School of Medicine, University of Girona, Girona, Spain
Universal Sequencing Technology Corp., Canton, MA
BioRxiv preprint
DOI: 10.1101/2023.03.05.531179