1,720,978 research outputs found
TAIL-seq: Genome-wide determination of poly(A) tail length and 3' end modifications
No full textGlobal investigation of the 30 extremity of mRNA (30-terminome), despite its importance in gene regulation,has not been feasible due to technical challenges associated with homopolymeric sequences
and relative paucity of mRNA. We here develop a method, TAIL-seq, to sequence the very end of
mRNA molecules. TAIL-seq allows us to measure poly(A) tail length at the genomic scale. Median poly(A) length is 50–100 nt in HeLa and NIH 3T3 cells. Poly(A) length correlates with mRNA half-life,
but not with translational efficiency. Surprisingly, we discover widespread uridylation and guanylation at the downstream of poly(A) tail. The U tails are generally attached to short poly(A) tails (40 nt), implicating their generic roles in mRNA stability control. TAIL-seq is a potent tool to dissect dynamic control of mRNA turnover and translational control, and to discover unforeseen features of RNA cleavage and tailing.11011101sciescopu
Regulation of Poly(A) Tail and Translation during the Somatic Cell Cycle
No full textPoly(A) tails are critical for mRNA stability and translation. However, recent studies have challenged this view, showing that poly(A) tail length and translation efficiency are decoupled in non-embryonic cells. Using TAIL-seq and ribosome profiling, we investigate poly(A) tail dynamics and translational control in the somatic cell cycle. We find dramatic changes in poly(A) tail lengths of cell-cycle regulatory genes like CDK1, TOP2A, and FBXO5, explaining their translational repression in M phase. We also find that poly(A) tail length is coupled to translation when the poly(A) tail is 20 nucleotide poly(A) tails, their translation is regulated mainly via poly(A) tail length-independent mechanisms during the cell cycle. Specifically, we find that terminal oligopyrimidine (TOP) tract-containing transcripts escape global translational suppression in M phase and are actively translated. Our quantitative and comprehensive data provide a revised view of translational control in the somatic cell cycle. © 2016 Elsevier Inc.128321sciescopu
MTAIL-seq reveals dynamic poly(A) tail regulation in oocyte-to-embryo development
No full textEukaryotic mRNAs are subject to multiple types of tailing that critically influence mRNA stability and translatability. To investigate RNA tails at the genomic scale, we previously developed TAIL-seq, but its low sensitivity precluded its application to biological materials of minute quantity. In this study, we report a new version of TAIL-seq (mRNA TAIL-seq [mTAIL-seq]) with enhanced sequencing depth for mRNAs (by ~1000-fold compared with the previous version). The improved method allows us to investigate the regulation of poly(A) tails in Drosophila oocytes and embryos. We found that maternal mRNAs are polyadenylated mainly during late oogenesis, prior to fertilization, and that further modulation occurs upon egg activation. Wispy, a noncanonical poly(A) polymerase, adenylates the vast majority of maternal mRNAs, with a few intriguing exceptions such as ribosomal protein transcripts. By comparing mTAIL-seq data with ribosome profiling data, we found a strong coupling between poly(A) tail length and translational efficiency during egg activation. Our data suggest that regulation of poly(A) tails in oocytes shapes the translatomic landscape of embryos, thereby directing the onset of animal development. By virtue of the high sensitivity, low cost, technical robustness, and broad accessibility, mTAIL-seq will be a potent tool to improve our understanding of mRNA tailing in diverse biological systems. © 2016 Lim et al122221sciescopu
TUT7 controls the fate of precursor microRNAs by using three different uridylation mechanisms
No full textTerminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis. Using biochemistry, single-molecule, and deep sequencing techniques, we here investigate the mechanism by which human TUT7 (also known as ZCCHC6) recognizes and uridylates precursor miRNAs (pre-miRNAs) in the absence of Lin28. We find that the overhang of a pre-miRNA is the key structural element that is recognized by TUT7 and its paralogues, TUT4 (ZCCHC11) and TUT2 (GLD2/PAPD4). For group II pre-miRNAs, which have a 1-nt 3′ overhang, TUT7 restores the canonical end structure (2-nt 3′ overhang) through mono-uridylation, thereby promoting miRNA biogenesis. For pre-miRNAs where the 3′ end is further recessed into the stem (as in 3′ trimmed pre-miRNAs), TUT7 generates an oligo-U tail that leads to degradation. In contrast to Lin28-stimulated oligo-uridylation, which is processive, a distributive mode is employed by TUT7 for both mono- and oligo-uridylation in the absence of Lin28. The overhang length dictates the frequency (but not duration) of the TUT7-RNA interaction, thus explaining how TUT7 differentiates pre-miRNA species with different overhangs. Our study reveals dual roles and mechanisms of uridylation in repair and removal of defective pre-miRNAs. Synopsis Uridylation of miRNA precursors can either stimulate processing or trigger RNA degradation. This study shows how RNA overhang structure and the mode of TUTase binding facilitate differential uridylation of specific precursor types. Terminal uridylyl transferases (TUTs) exert multiple roles in miRNA biogenesis by uridylating precursor miRNAs, thereby determining their fates. For group II pre-miRNAs, which carry a 1-nt 3′ overhang, TUT7/4/2 restore the canonical end structure through mono-uridylation, promoting miRNA biogenesis. For 3′ trimmed pre-miRNAs, TUT7/4 distributively generate an oligo-U tail that triggers degradation. TUT7 distinguishes pre-miRNA species at the binding step and shows distinct binding frequency to different RNA substrates. Uridylation of miRNA precursors can either stimulate processing or trigger RNA degradation. This study shows how RNA overhang structure and the mode of TUTase binding facilitate differential uridylation of specific precursor types. © 2015 The Authors. Published under the terms of the CC BY 4.0 license122251sciescopu
Partitioning RNAs by length improves transcriptome reconstruction from short-read RNA-seq data.
No full textThe accuracy of methods for assembling transcripts from short-read RNA sequencing data is limited by the lack of long-range information. Here we introduce Ladder-seq, an approach that separates transcripts according to their lengths before sequencing and uses the additional information to improve the quantification and assembly of transcripts. Using simulated data, we show that a kallisto algorithm extended to process Ladder-seq data quantifies transcripts of complex genes with substantially higher accuracy than conventional kallisto. For reference-based assembly, a tailored scheme based on the StringTie2 algorithm reconstructs a single transcript with 30.8% higher precision than its conventional counterpart and is more than 30% more sensitive for complex genes. For de novo assembly, a similar scheme based on the Trinity algorithm correctly assembles 78% more transcripts than conventional Trinity while improving precision by 78%. In experimental data, Ladder-seq reveals 40% more genes harboring isoform switches compared to conventional RNA sequencing and unveils widespread changes in isoform usage upon m6A depletion by Mettl14 knockout
Next-generation libraries for robust RNA interference-based genome-wide screens
No full textGenetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity131351sciescopu
Temporal Landscape of MicroRNA-Mediated Host-Virus Crosstalk during Productive Human Cytomegalovirus Infection
No full textTemporal profiles of miRNA activity during productive
virus infection can provide fundamental insights
into host-virus interactions. Most reported miRNA
targetome analyses in the context of virus infection
have been performed in latently infected cells and
lack reliable models for quantifying the suppression
efficacy at specific miRNA target sites. Here, we
identified highly competent temporal miRNA targetomes
during lytic HCMV infection by using AGOCLIP-
seq together with a bioinformatic method that
quantifies miRNA functionality at a specific target
site, called ACE-scoring. The repression efficiency
at target sites correlates with the magnitude of the
ACE-score, and temporal HCMV-encoded miRNA
targetomes identified by ACE-scoring were significantly
enriched in functional categories involved in
pathways central for HCMV biology. Furthermore,
comparative analysis between human and viral
miRNA targetomes supports the existence of intimate
cooperation and co-targeting between them.
Our holistic survey provides a valuable resource for
understanding host-virus interactions during lytic
HCMV infection. ⓒ2015 Elsevier Inc.11091sciescopu
Uridylation by TUT4 and TUT7 marks mRNA for degradation
No full textUridylation occurs pervasively on mRNAs, yet its
mechanism and significance remain unknown. By
applying TAIL-seq, we identify TUT4 and TUT7
(TUT4/7), also known as ZCCHC11 and ZCCHC6,
respectively, as mRNA uridylation enzymes. Uridylation
readily occurs on deadenylated mRNAs in cells.
Consistently, purified TUT4/7 selectively recognize
and uridylate RNAs with short A-tails (less than
25 nt) in vitro. PABPC1 antagonizes uridylation of
polyadenylated mRNAs, contributing to the specificity
for short A-tails. In cells depleted of TUT4/7,
the vast majority of mRNAs lose the oligo-U-tails,
and their half-lives are extended. Suppression of
mRNA decay factors leads to the accumulation of
oligo-uridylated mRNAs. In line with this, microRNA
induces uridylation of its targets, and TUT4/7 are
required for enhanced decay of microRNA targets.
Our study explains the mechanism underlying
selective uridylation of deadenylated mRNAs and
demonstrates a fundamental role of oligo-U-tail as
a molecular mark for global mRNA decay.170701sciescopu
Analyzing viral epitranscriptomes using nanopore direct RNA sequencing
No full textRNA modifications are a common occurrence across all domains of life. Several chemical modifications, including N(6)-methyladenosine, have also been found in viral transcripts and viral RNA genomes. Some of the modifications increase the viral replication efficiency while also helping the virus to evade the host immune system. Nonetheless, there are numerous examples in which the host's RNA modification enzymes function as antiviral factors. Although established methods like MeRIP-seq and miCLIP can provide a transcriptome- wide overview of how viral RNA is modified, it is difficult to distinguish between the complex overlapping viral transcript isoforms using the short read-based techniques. Nanopore direct RNA sequencing (DRS) provides both long reads and direct signal readings, which may carry information about the modifications. Here, we describe a refined protocol for analyzing the RNA modifications in viral transcriptomes using nanopore technology. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplemental material for this article may be found at 10.1007/s12275-022-2324-4
LIN28A Is a Suppressor of ER-Associated Translation in Embryonic Stem Cells
No full textLIN28 plays a critical role in developmental transition, glucose metabolism, and tumorigenesis. At the molecular level, LIN28 is known to repress maturation of let-7 microRNAs and enhance translation of certain mRNAs. In this study, we obtain a genome-wide view of the molecular function of LIN28A in mouse embryonic stem cells by carrying out RNA crosslinking-immunoprecipitation-sequencing (CLIP-seq) and ribosome footprinting. We find that, in addition to let-7 precursors, LIN28A binds to a large number of spliced mRNAs. LIN28A recognizes AAGNNG, AAGNG, and less frequently UGUG, which are located in the terminal loop of a small hairpin. LIN28A is localized to the periendo-plasmic reticulum (ER) area and inhibits translation of mRNAs that are destined for the ER, reducing the synthesis of transmembrane proteins, ER or Golgi lumen proteins, and secretory proteins. Our study suggests a selective regulatory mechanism for ER-associated translation and reveals an unexpected role of LIN28A as a global suppressor of genes in the secretory pathway.11211111sciescopu
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