Non-sequential and multi-step splicing of the dystrophin transcript.
Isabella Gazzoli,Irina Pulyakhina,Nisha Verwey,Yavuz Ariyurek,Jeroen F.J. Laros,Peter A C 't Hoen,Annemieke Aartsma-Rus +6 more
TLDR
Overall, the data show for the first time in a human transcript, that multi-step intron removal is a general feature of mRNA splicing.Abstract:
The dystrophin protein encoding DMD gene is the longest human gene. The 2.2 Mb long human dystrophin transcript takes 16 hours to be transcribed and is co-transcriptionally spliced. It contains long introns (24 over 10kb long, 5 over 100kb long) and the heterogeneity in intron size makes it an ideal transcript to study different aspects of the human splicing process. Splicing is a complex process and much is unknown regarding the splicing of long introns in human genes. Here, we used ultra-deep transcript sequencing to characterize splicing of the dystrophin transcripts in 3 different human skeletal muscle cell lines, and explored the order of intron removal and multi-step splicing. Coverage and read pair analyses showed that around 40% of the introns were not always removed sequentially. Additionally, for the first time, we report that non-consecutive intron removal resulted in 3 or more joined exons which are flanked by unspliced introns and we defined these joined exons as an exon block. Lastly, computational and experimental data revealed that, for the majority of dystrophin introns, multistep splicing events are used to splice out a single intron. Overall, our data show for the first time in a human transcript, that multi-step intron removal is a general feature of mRNA splicing.read more
Citations
More filters
Journal ArticleDOI
RNA sequencing: the teenage years
TL;DR: Advances in RNA-sequencing technologies and methods over the past decade are discussed and adaptations that are enabling a fuller understanding of RNA biology are outlined, from when and where an RNA is expressed to the structures it adopts.
Journal ArticleDOI
Deep intronic mutations and human disease.
TL;DR: Evidence from mRNA analysis and entire genomic sequencing indicates that pathogenic mutations can occur deep within the introns of over 75 disease-associated genes, highlighting the importance of studying variation in deep intronic sequence as a cause of monogenic disorders as well as hereditary cancer syndromes.
Journal ArticleDOI
Dynamic imaging of nascent RNA reveals general principles of transcription dynamics and stochastic splice site selection.
Yihan Wan,Dimitrios G. Anastasakis,Joseph Rodriguez,Murali Palangat,Prabhakar R. Gudla,George Zaki,Mayank Tandon,Gianluca Pegoraro,Carson C. Chow,Markus Hafner,Daniel R. Larson +10 more
TL;DR: In this paper, a quasi-genome-scale platform for observing synthesis and processing kinetics of single nascent RNA molecules in real time is established, and all observed genes show transcriptional bursting.
Journal ArticleDOI
Multiple Exon Skipping in the Duchenne Muscular Dystrophy Hot Spots: Prospects and Challenges
TL;DR: An update on DMD genotype-phenotype associations using a global DMD database is provided and the rationale for multiple exon skipping development is provided, particularly for exons 45–55 skipping and an emerging therapeutic concept, exons 3–9 skipping.
Journal ArticleDOI
The determinants of alternative RNA splicing in human cells
TL;DR: It is concluded that the final structure of RNA is pre-determined by a complex interplay between cis- and trans-acting factors.
References
More filters
Journal ArticleDOI
The Sequence Alignment/Map format and SAMtools
Heng Li,Bob Handsaker,Alec Wysoker,T. J. Fennell,Jue Ruan,Nils Homer,Gabor T. Marth,Gonçalo R. Abecasis,Richard Durbin +8 more
TL;DR: SAMtools as discussed by the authors implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments.
Journal ArticleDOI
Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data
Jan M. Ruijter,Christian Ramakers,W. M. H. Hoogaars,Yann Karlen,O. Bakker,M. J. B. Van Den Hoff,Antoon F.M. Moorman +6 more
TL;DR: This article showed that baseline estimation errors are directly reflected in the observed PCR efficiency values and are thus propagated exponentially in the estimated starting concentrations as well as 'fold-difference' results.
Journal ArticleDOI
The Spliceosome: Design Principles of a Dynamic RNP Machine
TL;DR: The spliceosome exhibits exceptional compositional and structural dynamics that are exploited during substrate-dependent complex assembly, catalytic activation, and active site remodeling in the pre-mRNAs.
Journal ArticleDOI
Fast and SNP-tolerant detection of complex variants and splicing in short reads
Thomas D. Wu,Serban Nacu +1 more
TL;DR: Computational methods for fast detection of complex variants and splicing in short reads, based on a successively constrained search process of merging and filtering position lists from a genomic index are presented.
Journal ArticleDOI
Solution Hybrid Selection with Ultra-long Oligonucleotides for Massively Parallel Targeted Sequencing
Andreas Gnirke,Alexandre Melnikov,Jared Maguire,Peter Rogov,Emily M LeProust,William Brockman,William Brockman,Timothy Fennell,Georgia Giannoukos,Sheila Fisher,Carsten Russ,Stacey Gabriel,David B. Jaffe,Eric S. Lander,Eric S. Lander,Eric S. Lander,Chad Nusbaum +16 more
TL;DR: A capture method that uses biotinylated RNA 'baits' to fish targets out of a 'pond' of DNA fragments that uniformity was such that ∼60% of target bases in the exonic 'catch', and ∼80% in the regional catch, had at least half the mean coverage.