Biology subjects

Pelechano, V.

Publications and source records attributed to Pelechano, V..

24 records · Page 2Linked to original sources

bioRxiv2020

Fivepseq: analysis of 5' count distribution in RNA-seq datasets for inference on mRNA degradation and translation

In eukaryotes, 5-3 co-translation degradation machinery follows the last translating ribosome providing an in vivo footprint of its position. Thus 5P degradome sequencing, in addition to informing about RNA decay, also provides valuable information regarding ribosome dynamics. Multiple experimental methods have been developed to investigate the mRNA degradome, however computational tools for their reproducible analysis are lacking. Here we present fivepseq: an easy-to-use application for analysis and interactive visualization of 5P degradome data. This tool performs both metagene and gene specific analysis, and allows to easily investigate codon specific ribosome pauses. To demonstrate its ability to provide new biological information, we investigate gene specific ribosome pauses in S. cerevisiae after eIF5A depletion. In addition to identifying pauses at expected codon motifs, we identify multiple genes with strain-specific frameshifts. To show its wide applicability, we investigate more complex 5P degradome from A. thaliana and discover both motif-specific ribosome protection associated with particular developmental stages, as well as generally increased ribosome protection at termination level associated with age. Our work shows how the use of improved analysis tools for the study of 5P degradome can significantly increase the biological information that can be derived from such datasets and facilitate its reproducible analysis. KEY POINTSO_LIAnalysis of 5P degradome data with fivepseq informs about global and gene-specific translational features. C_LIO_LIFrameshifts in translation-related genes in S. cerevisiae may be linked to ribosome stalling. C_LIO_LIRibosome protection at termination and codon motifs are linked to development in A. Thaliana. C_LI

genomics↗

bioRxiv2019

Improved transcriptome annotation and read-through transcript identification with TIF-Seq2

Eukaryotic transcriptomes are complex involving thousands of overlapping transcripts. The interleaved nature of the transcriptome limits our ability to identify regulatory regions and, in some cases, can lead to misinterpretation of gene expression. To improve the understanding of the overlapping transcriptome, we have developed an optimized method, TIF-Seq2, able to sequence simultaneously the 5 and 3 ends of individual RNA molecules at single-nucleotide resolution. We investigated the transcriptome of a well characterized human cell line (K562) and identify thousands of unannotated transcript isoforms. By focusing on transcripts which are challenging to be investigated with RNA-seq, we accurately defined boundaries of lowly expressed unannotated and read-though transcripts putatively encoding fusion genes. We validated our results by targeted long-read sequencing and standard RNA-Seq for chronic myeloid leukaemia patient samples. Taking the advantage of TIF-Seq2, we explore transcription regulation among the overlapping units and investigate their crosstalk. We show that most overlapping upstream transcripts use poly(A) sites within the first 2 kb of the downstream transcription unit. Our work shows that, by paring the 5 and 3 end of each RNA, TIF-Seq2 can improve the annotation of complex genomes, facilitates accurate assignment of promoters to genes and easily identify transcriptionally fused genes. Key pointsO_LIStudy of TSS-PAS co-occurrence allows dissecting complex overlapping transcription units. C_LIO_LIPartially overlapping transcription units in human commonly use PAS within the first 2Kb. C_LIO_LITIF-Seq2 facilitates the identification of lowly expressed and transcriptionally fused genes. C_LI

genomics↗

bioRxiv2019

Transcript isoform sequencing reveals widespread promoter-proximal transcriptional termination

Higher organisms achieve optimal gene expression by tightly regulating the transcriptional activity of RNA Polymerase II (RNAPII) along DNA sequences of genes1. RNAPII density across genomes is typically highest where two key choices for transcription occur: near transcription start sites (TSSs) and polyadenylation sites (PASs) at the beginning and end of genes, respectively2,3. Alternative TSSs and PASs amplify the number of transcript isoforms from genes4, but how alternative TSSs connect to variable PASs is unresolved from common transcriptomics methods. Here, we define TSS/PAS pairs for individual transcripts in Arabidopsis thaliana using an improved Transcript Isoform sequencing (TIF-seq) protocol and find on average over four different isoforms corresponding to variable TSS/PAS pairs per expressed gene. While intragenic initiation represents a large source of regulated isoform diversity, we discover that [~] 14% of expressed genes generate relatively unstable short promoter-proximal RNAs (sppRNAs) from nascent transcript cleavage and polyadenylation shortly after initiation. The location of sppRNAs coincides with increased RNAPII density, indicating these large pools of promoter-stalled RNAPII across genomes are often engaged in transcriptional termination. RNAPII elongation factors progress transcription beyond sites of sppRNA formation, demonstrating RNAPII density near promoters represents a checkpoint for early transcriptional termination that governs full-length gene isoform expression.

genomics↗

bioRxiv2018

Chromatin-dependent cryptic promoters encode alternative protein isoforms in yeast.

Cryptic transcription is widespread and generates a heterogeneous group of RNA molecules of unknown function. To improve our understanding of cryptic transcription, we investigated their transcription start site usage, chromatin organization and post-transcriptional consequences in Saccharomyces cerevisiae. We show that transcription start sites (TSSs) of chromatin-sensitive internal cryptic transcripts retain comparable features of canonical TSSs in terms of DNA sequence, directionality and chromatin accessibility. We degine the 5 and 3 boundaries of cryptic transcripts and show that, contrary to RNA degradation-sensitive ones, they often overlap with the end of the gene thereby using the canonical polyadenylation site and associate to polyribosomes. We show that chromatin-sensitive cryptic transcripts can be recognized by ribosomes and may produce truncated polypeptides from downstream, in-frame start codons. Finally, we congirm the presence of the predicted polypeptides by reanalyzing N-terminal proteomic datasets. Our work suggests that a fraction of chromatin-sensitive internal cryptic promoters are in fact alternative truncated mRNA isoforms. The expression of these chromatin-sensitive isoforms is conserved from yeast to human expanding the functional consequences of cryptic transcription and proteome complexity.

genomics↗

bioRxiv2018

Understanding RNP remodelling uncovers RBPs functionally required for viral replication

The compendium of RNA-binding proteins (RBPome) has been greatly expanded by the development of RNA-interactome capture (RNA-IC). However, it remains unknown how responsive is the RBPome and whether these responses are biologically relevant. To answer these questions, we created comparative RNA-IC to analyse cells challenged with an RNA virus, called sindbis (SINV). Strikingly, the virus altered the activity of 245 RBPs, many of which were newly discovered by RNA-IC. Mechanistically, alterations in RNA binding upon SINV infection are caused by changes in the subcellular localisation of RBPs and RNA availability. Moreover, RBPome responses are crucial, as perturbation of dynamic RBPs modulates the capacity of the virus to infect the cell. For example, ablation of XRN1 causes cells to be refractory to infection, while GEMIN5 moonlights as a novel antiviral factor. Therefore, RBPome remodelling provides a mechanism by which cells can extensively rewire gene expression in response to physiological cues.\n\nHIGHLIGHTSO_LIA quarter of the RBPome remodels upon SINV infection.\nC_LIO_LIThe remodelling is caused by changes in protein localisation and RNA availability.\nC_LIO_LIRewiring of the RBPome is crucial for viral infection efficacy.\nC_LIO_LIWe discover RBPs with previously unknown anti- or pro-viral activity.\nC_LI

molecular biology↗

bioRxiv2018

Transcription-driven Chromatin Repression of Intragenic Promoters

Progression of RNA polymerase II (RNAPII) transcription relies on the appropriately positioned activities of elongation factors. The resulting profile of factors and chromatin signatures along transcription units provides a \"positional information system\" for transcribing RNAPII. Here, we investigate a chromatin-based mechanism that suppresses intragenic initiation of RNAPII transcription. We demonstrate that RNAPII transcription across gene promoters represses their function in plants. This repression is characterized by reduced promoter-specific molecular signatures and increased molecular signatures associated with RNAPII elongation. The FACT histone chaperone complex is required for this repression mechanism. Genome-wide mapping of Transcription Start Sites (TSSs) reveals thousands of discrete intragenic TSS positions in FACT mutants. Histone 3 lysine 4 mono-methylation poises exonic sites to initiate RNAPII transcription in FACT mutants. Uncovering the mechanism for intragenic TSS repression through the act of RNAPII elongation has important implications for understanding pervasive RNAPII transcription and the regulation of transcript isoform diversity.

genomics↗