Rnalib: a Python library for custom transcriptomics analyses.

Motivation: The efficient and reproducible analysis of high-throughput sequencing datasets necessitates the development of methodical and robust computational pipelines that integrate established and bespoke bioinformatics analysis tools, often written in high-level programming languages such as Python. Despite the increasing availability of programming libraries for genomics, there is a noticeable lack of tools specifically focused on transcriptomics. Key tasks in this area include the association of gene features (e.

Proteome-scale tagging and functional screening in mammalian cells by ORFtag.

The systematic determination of protein function is a key goal of modern biology, but remains challenging with current approaches. Here we present ORFtag, a versatile, cost-effective and highly efficient method for the massively parallel tagging and functional interrogation of proteins at the proteome scale. ORFtag uses retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous open reading frames (ORFs).

Splice_sim: a nucleotide conversion-enabled RNA-seq simulation and evaluation framework.

Nucleotide conversion RNA sequencing techniques interrogate chemical RNA modifications in cellular transcripts, resulting in mismatch-containing reads. Biases in mapping the resulting reads to reference genomes remain poorly understood. We present splice_sim, a splice-aware RNA-seq simulation and evaluation pipeline that introduces user-defined nucleotide conversions at set frequencies, creates mixture models of converted and unconverted reads, and calculates mapping accuracies per genomic annotation.

Mime-seq 2.0: a method to sequence microRNAs from specific mouse cell types.

Many microRNAs (miRNAs) are expressed with high spatiotemporal specificity during organismal development, with some being limited to rare cell types, often embedded in complex tissues. Yet, most miRNA profiling efforts remain at the tissue and organ levels. To overcome challenges in accessing the microRNomes from tissue-embedded cells, we had previously developed mime-seq (miRNome by methylation-dependent sequencing), a technique in which cell-specific miRNA methylation in C.

SLAMseq resolves the kinetics of maternal and zygotic gene expression during early zebrafish embryogenesis.

The maternal-to-zygotic transition (MZT) is a key developmental process in metazoan embryos that involves the activation of zygotic transcription (ZGA) and degradation of maternal transcripts. We employed metabolic mRNA sequencing (SLAMseq) to deconvolute the compound embryonic transcriptome in zebrafish. While mitochondrial zygotic transcripts prevail prior to MZT, we uncover the spurious transcription of hundreds of short and intron-poor genes as early as the 2-cell stage.

Hijacking of transcriptional condensates by endogenous retroviruses.

Most endogenous retroviruses (ERVs) in mammals are incapable of retrotransposition; therefore, why ERV derepression is associated with lethality during early development has been a mystery. Here, we report that rapid and selective degradation of the heterochromatin adapter protein TRIM28 triggers dissociation of transcriptional condensates from loci encoding super-enhancer (SE)-driven pluripotency genes and their association with transcribed ERV loci in murine embryonic stem cells. Knockdown of ERV RNAs or forced expression of SE-enriched transcription factors rescued condensate localization at SEs in TRIM28-degraded cells.

NMD is required for timely cell fate transitions by fine-tuning gene expression and regulating translation.

Cell fate transitions depend on balanced rewiring of transcription and translation programs to mediate ordered developmental progression. Components of the nonsense-mediated mRNA decay (NMD) pathway have been implicated in regulating embryonic stem cell (ESC) differentiation, but the exact mechanism is unclear. Here we show that NMD controls expression levels of the translation initiation factor and its premature termination codon-encoding isoform ( ).

Transcriptome-Wide Profiling of RNA Stability.

Gene expression is controlled at multiple levels, including RNA transcription and turnover. But determining the relative contributions of RNA biogenesis and decay to the steady-state abundance of cellular transcripts remains challenging because conventional transcriptomics approaches do not provide the temporal resolution to derive the kinetic parameters underlying steady-state gene expression.Here, we describe a protocol that combines metabolic RNA labeling by 4-thiouridine with chemical nucleoside conversion and whole-transcriptome sequencing followed by bioinformatics analysis to determine RNA stability in cultured cells at a genomic scale.

Systematic refinement of gene annotations by parsing mRNA 3' end sequencing datasets.

Alternative cleavage and polyadenylation generates mRNA 3' isoforms in a cell type-specific manner. Due to finite available RNA sequencing data of organisms with vast cell type complexity, currently available gene annotation resources are incomplete, which poses significant challenges to the comprehensive interpretation and quantification of transcriptomes. In this chapter, we introduce 3'GAmES, a stand-alone computational pipeline for the identification and quantification of novel mRNA 3'end isoforms from 3'mRNA sequencing data.

Structure-function analysis of microRNA 3'-end trimming by Nibbler.

Nibbler (Nbr) is a 3'-to-5' exoribonuclease whose catalytic 3'-end trimming activity impacts microRNA (miRNA) and PIWI-interacting RNA (piRNA) biogenesis. Here, we report on structural and functional studies to decipher the contributions of Nbr's N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3'-end trimming. We have solved the crystal structures of the NTD core and EXO domains of Nbr, both in the apo-state.

Conformation of sister chromatids in the replicated human genome.

The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C) analysis has revealed a complex genomic landscape of internal chromosomal structures in vertebrate cells, but the identical sequence of sister chromatids has made it difficult to determine how they topologically interact in replicated chromosomes. Here we describe sister-chromatid-sensitive Hi-C (scsHi-C), which is based on labelling of nascent DNA with 4-thio-thymidine and nucleoside conversion chemistry.

Determining mRNA Stability by Metabolic RNA Labeling and Chemical Nucleoside Conversion.

The varying rates at which mRNAs decay are tightly coordinated with transcriptional changes to shape gene expression during development and disease. But currently available RNA sequencing approaches lack the temporal information to determine the relative contribution of RNA biogenesis, processing and turnover to the establishment of steady-state gene expression profiles.Here, we describe a protocol that combines metabolic RNA labeling with chemical nucleoside conversion by thiol-linked alkylation of 4-thiouridine to determine RNA stability in cultured cells (SLAMseq).

Time-Resolved Small RNA Sequencing Unravels the Molecular Principles of MicroRNA Homeostasis.

Argonaute-bound microRNAs silence mRNA expression in a dynamic and regulated manner to control organismal development, physiology, and disease. We employed metabolic small RNA sequencing for a comprehensive view on intracellular microRNA kinetics in Drosophila. Based on absolute rate of biogenesis and decay, microRNAs rank among the fastest produced and longest-lived cellular transcripts, disposing up to 10 copies per cell at steady-state.

Publisher Correction: Sequencing cell-type-specific transcriptomes with SLAM-ITseq.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Sequencing cell-type-specific transcriptomes with SLAM-ITseq.

Analysis of cell-type-specific transcriptomes is vital for understanding the biology of tissues and organs in the context of multicellular organisms. In this Protocol Extension, we combine a previously developed cell-type-specific metabolic RNA labeling method (thiouracil (TU) tagging) and a pipeline to detect the labeled transcripts by a novel RNA sequencing (RNA-seq) method, SLAMseq (thiol (SH)-linked alkylation for the metabolic sequencing of RNA). By injecting a uracil analog, 4-thiouracil, into transgenic mice that express cell-type-specific uracil phosphoribosyltransferase (UPRT), an enzyme required for 4-thiouracil incorporation into newly synthesized RNA, only cells expressing UPRT synthesize thiol-containing RNA.

Quantification of experimentally induced nucleotide conversions in high-throughput sequencing datasets.

Background: Methods to read out naturally occurring or experimentally introduced nucleic acid modifications are emerging as powerful tools to study dynamic cellular processes. The recovery, quantification and interpretation of such events in high-throughput sequencing datasets demands specialized bioinformatics approaches.

Results: Here, we present Digital Unmasking of Nucleotide conversions in K-mers (DUNK), a data analysis pipeline enabling the quantification of nucleotide conversions in high-throughput sequencing datasets.

Structural basis for acceptor RNA substrate selectivity of the 3' terminal uridylyl transferase Tailor.

Non-templated 3'-uridylation of RNAs has emerged as an important mechanism for regulating the processing, stability and biological function of eukaryotic transcripts. In Drosophila, oligouridine tailing by the terminal uridylyl transferase (TUTase) Tailor of numerous RNAs induces their degradation by the exonuclease Dis3L2, which serves functional roles in RNA surveillance and mirtron RNA biogenesis. Tailor preferentially uridylates RNAs terminating in guanosine or uridine nucleotides but the structural basis underpinning its RNA substrate selectivity is unknown.

Small RNAs Are Trafficked from the Epididymis to Developing Mammalian Sperm.

The biogenesis of the RNA payload of mature sperm is of great interest, because RNAs delivered to the zygote at fertilization can affect early development. Here, we tested the hypothesis that small RNAs are trafficked to mammalian sperm during the process of post-testicular maturation in the epididymis. By characterizing small RNA dynamics during germ cell maturation in mice, we confirm and extend prior observations that sperm undergo a dramatic switch in the RNA payload from piRNAs to tRNA fragments (tRFs) upon exiting the testis and entering the epididymis.

Analysis of 3' End Modifications in microRNAs by High-Throughput Sequencing.

MicroRNAs are ~22 nt small, non-coding RNAs that direct posttranscriptional silencing of gene expression to regulate animal development, physiology, and disease. An emerging mechanism that controls the biogenesis of microRNAs is the addition of non-templated nucleotides, predominantly uridine, to the 3' end of precursor-microRNAs, in a process that is commonly referred to as tailing. Here, we describe methods that enable the systematic characterization of tailing events in mature microRNAs and their precursors.

SLAM-ITseq: sequencing cell type-specific transcriptomes without cell sorting.

Cell type-specific transcriptome analysis is an essential tool for understanding biological processes in which diverse types of cells are involved. Although cell isolation methods such as fluorescence-activated cell sorting (FACS) in combination with transcriptome analysis have widely been used so far, their time-consuming and harsh procedures limit their applications. Here, we report a novel metabolic RNA sequencing method, SLAM-ITseq, which metabolically labels RNA with 4-thiouracil in a specific cell type followed by detection through an RNA-seq-based method that specifically distinguishes the thiolated uridine by base conversion.

SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis.

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment.

Cell-type specific sequencing of microRNAs from complex animal tissues.

MicroRNAs (miRNAs) play an essential role in the post-transcriptional regulation of animal development and physiology. However, in vivo studies aimed at linking miRNA function to the biology of distinct cell types within complex tissues remain challenging, partly because in vivo miRNA-profiling methods lack cellular resolution. We report microRNome by methylation-dependent sequencing (mime-seq), an in vivo enzymatic small-RNA-tagging approach that enables high-throughput sequencing of tissue- and cell-type-specific miRNAs in animals.

Thiol-linked alkylation of RNA to assess expression dynamics.

Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM seq), an orthogonal-chemistry-based RNA sequencing technology that detects 4-thiouridine (sU) incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM seq enabled rapid access to RNA-polymerase-II-dependent gene expression dynamics in the context of total RNA.

Genetic and mechanistic diversity of piRNA 3'-end formation.

Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and therefore have important roles in eukaryotic gene silencing. Of the three small RNA classes, microRNAs and short interfering RNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. PIWI-interacting RNAs (piRNAs)-the 22-30-nt-long guides for PIWI-clade Ago proteins that silence transposons in animal gonads-are generated independently of Dicer from single-stranded precursors.

Molecular basis for cytoplasmic RNA surveillance by uridylation-triggered decay in Drosophila.

The posttranscriptional addition of nucleotides to the 3' end of RNA regulates the maturation, function, and stability of RNA species in all domains of life. Here, we show that in flies, 3' terminal RNA uridylation triggers the processive, 3'-to-5' exoribonucleolytic decay via the RNase II/R enzyme CG16940, a homolog of the human Perlman syndrome exoribonuclease Dis3l2. Together with the TUTase Tailor, dmDis3l2 forms the cytoplasmic, terminal RNA uridylation-mediated processing (TRUMP) complex that functionally cooperates in the degradation of structured RNA RNA immunoprecipitation and high-throughput sequencing reveals a variety of TRUMP complex substrates, including abundant non-coding RNA, such as 5S rRNA, tRNA, snRNA, snoRNA, and the essential RNase MRP Based on genetic and biochemical evidence, we propose a key function of the TRUMP complex in the cytoplasmic quality control of RNA polymerase III transcripts.