Biology subjects

Kim, V. N.

Publications and source records attributed to Kim, V. N..

6 recordsLinked to original sources

bioRxiv2023

The Role of Terminal Uridyl Transferases in the Circadian Rhythm

The 3 terminal oligo-uridylation, a post-transcriptional mRNA modification, is conserved among eukaryotes and drives mRNA degradation, thereby affecting several key biological processes such as animal development and viral infection. Our TAIL-seq experiment of mouse liver mRNA collected from six zeitgeber times reveals transcripts with rhythmic poly(A) tail lengths and demonstrates that overall 3 terminal uridylation frequencies at mRNA poly(A) tail very-ends undergo rhythmic change. Consistently, major terminal uridylyl transferases, TUT4 and TUT7, have cycling protein expression in mouse liver corresponding to 3 terminal uridylation rhythms, indicating that the cycling expression of TUTases correlates with the rhythmic pattern of uridylation. Furthermore, the double knockdown of TUT4 and TUT7 in U2OS cells lengthens the circadian period and decreases the rhythmic amplitude of clock gene expression. Our work thoroughly profiles the dynamic changes in poly(A) tail lengths and terminal modifications and uncovers uridylation as a post-transcriptional modulator in the mammalian circadian clock.

molecular biology↗

bioRxiv2020

The SARS-CoV-2 RNA interactome

SARS-CoV-2 is an RNA virus whose success as a pathogen relies on its ability to repurpose host RNA-binding proteins (RBPs) to form its own RNA interactome. Here, we developed and applied a robust ribonucleoprotein capture protocol to uncover the SARS-CoV-2 RNA interactome. We report 109 host factors that directly bind to SARS-CoV-2 RNAs including general antiviral factors such as ZC3HAV1, TRIM25, and PARP12. Applying RNP capture on another coronavirus HCoV-OC43 revealed evolutionarily conserved interactions between viral RNAs and host proteins. Network and transcriptome analyses delineated antiviral RBPs stimulated by JAK-STAT signaling and proviral RBPs responsible for hijacking multiple steps of the mRNA life cycle. By knockdown experiments, we further found that these viral-RNA-interacting RBPs act against or in favor of SARS-CoV-2. Overall, this study provides a comprehensive list of RBPs regulating coronaviral replication and opens new avenues for therapeutic interventions.

microbiology↗

bioRxiv2020

ERH as a component of the Microprocessor facilitates the maturation of suboptimal microRNAs

The Microprocessor complex cleaves the primary transcript of microRNA (pri-miRNA) to initiate miRNA maturation. Microprocessor is known to consist of RNase III DROSHA and dsRNA-binding DGCR8. Here we identify Enhancer of Rudimentary Homolog (ERH) as a new component of the Microprocessor. ERH binds to a conserved region in the N-terminus of DGCR8. Knockdown of ERH or deletion of the DGCR8 N-terminus results in a decrease of processing of primary miRNAs with suboptimal hairpin structures that reside in polycistronic miRNA clusters. ERH increases the processing of suboptimal pri-miR-451 in a manner dependent on its neighboring pri-miR-144. Thus, the ERH dimer may mediate "cluster assistance" in which the Microprocessor is loaded onto a poor substrate with help from a high-affinity substrate in the same cluster. Our study reveals a role of ERH in the miRNA pathway.

molecular biology↗

bioRxiv2020

MicroRNA arm switching regulated by uridylation

Strand selection is a critical step in microRNA (miRNA) biogenesis. Although the dominant strand may alter depending on cellular contexts, the molecular mechanism and physiological significance of such alternative strand selection (or "arm switching") remain elusive. Here we find mir-324 as one of the strongly regulated miRNAs by arm switching, and identify terminal uridylyl transferases TUT4 and TUT7 as the key regulators. Uridylation of pre-mir-324 by TUT4/7 re-positions DICER on the pre-miRNA and shifts the cleavage site. This alternative processing produces a duplex with a different terminus, from which the 3' strand (3p) is selected instead of the 5' strand (5p). In glioblastoma, the TUT4/7 and 3p levels are upregulated while the 5p level is reduced. Manipulation of the strand ratio is sufficient to impair glioblastoma cell proliferation. This study uncovers a role of uridylation as a molecular switch in alternative strand selection and implicates its therapeutic potential.

molecular biology↗

bioRxiv2020

The architecture of SARS-CoV-2 transcriptome

SARS-CoV-2 is a betacoronavirus that is responsible for the COVID-19 pandemic. The genome of SARS-CoV-2 was reported recently, but its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we here present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous recombination events, both canonical and noncanonical. In addition to the genomic RNA and subgenomic RNAs common in all coronaviruses, SARS-CoV-2 produces a large number of transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif being AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the internal modification and the 3' tail. Functional investigation of the unknown ORFs and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2. HighlightsO_LIWe provide a high-resolution map of SARS-CoV-2 transcriptome and epitranscriptome using nanopore direct RNA sequencing and DNA nanoball sequencing. C_LIO_LIThe transcriptome is highly complex owing to numerous recombination events, both canonical and noncanonical. C_LIO_LIIn addition to the genomic and subgenomic RNAs common in all coronaviruses, SARS-CoV-2 produces transcripts encoding unknown ORFs. C_LIO_LIWe discover at least 41 potential RNA modification sites with an AAGAA motif. C_LI

genomics↗

bioRxiv2018

AQ-seq: Accurate quantification of microRNAs and their variants

MicroRNAs (miRNAs) modulate diverse biological and pathological processes via post-transcriptional gene silencing. High-throughput small RNA sequencing (sRNA-seq) has been widely adopted to investigate the functions and regulatory mechanisms of miRNAs. However, accurate quantification has been limited owing to the severe ligation bias in conventional sRNA-seq methods. Here we present a high-throughput protocol, termed AQ-seq (accurate quantification by sequencing), that utilizes adapters with terminal degenerate sequences and a high concentration of polyethylene glycol (PEG), which removes the ligation bias during library preparation. By accurately measuring miRNAs and their variants (known as isomiRs), we identify alternatively processed miRNAs and correct the 5' end usage and strand preference that have been misannotated. We also uncover highly modified miRNAs that are uridylated and adenylated. Taken together, AQ-seq reveals the complexity of the miRNA isoform landscape, allowing us to refine miRNA annotation and to advance our understanding of miRNA regulation. Furthermore, AQ-seq can be adopted to improve other ligation-based sequencing methods including crosslinking-immunoprecipitation-sequencing (CLIP-seq) and ribosome profiling (Ribo-seq).

molecular biology↗