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Global donor and acceptor splicing site kinetics in human cells
No full textRNA splicing is an essential part of eukaryotic gene expression. Although the mechanism of splicing has been extensively studied in vitro, in vivo kinetics for the two-step splicing reaction remain poorly understood. Here, we combine transient transcriptome sequencing (TT-seq) and mathematical modeling to quantify RNA metabolic rates at donor and acceptor splice sites across the human genome. Splicing occurs in the range of minutes and is limited by the speed of RNA polymerase elongation. Splicing kinetics strongly depends on the position and nature of nucleotides flanking splice sites, and on structural interactions between unspliced RNA and small nuclear RNAs in spliceosomal intermediates. Finally, we introduce the \‘yield\’ of splicing as the efficiency of converting unspliced to spliced RNA and show that it is highest for mRNAs and independent of splicing kinetics. These results lead to quantitative models describing how splicing rates and yield are encoded in the human genome.Genes are portions of DNA that carry the instructions to build proteins. In particular, they are formed of segments called exons, which contain the protein-building information, and of non-coding segments known as introns. Exons and introns alternate within a gene. To create a given protein, the cell first uses an enzyme, Polymerase II, to copy the entire related gene – including introns and exons – into a molecule of ribonucleic acid, or RNA. As the gene is copied, a machine called the spliceosome comes onto the RNA molecule to remove the introns and create the final RNA template used to produce proteins. The spliceosome works by recognizing specific sequences that signal the border between introns and exons. Once the machine is bound to these ‘splice sites’ on each side of an intron, it brings the two neighboring exons close together and cuts out the intron. The two ends of the exons are then attached together. Previous studies have measured how fast introns are removed, but it remained unclear how long it takes to cut individual splice sites genome-wide. To address this question, Wachutka, Caizzi et al. combined a mathematical approach with a biochemical method that purifies newly made RNA in human cells. The experiments showed that it only took a few minutes to cut most splice sites. Cutting splice sites that bordered very long introns was slower, presumably because the Polymerase II took longer to produce these introns. In addition, the genetic sequences of the splice sites affected the time it took to remove the introns: some made it harder for the spliceosome to recognize where to cut, but others made it easier. Mistakes in removing introns from RNA can lead to producing abnormal proteins, and many diseases such as cystic fibrosis and Duchenne muscular dystrophy can be caused by such errors. In particular, small changes in the sequences at the splice sites or in the surrounding areas can create problems when it comes to eliminating introns. Decrypting the dynamics of intron cutting and removal may give scientists new insight into the molecular causes of cystic fibrosis and many other genetic disorders.RNA splicing is an essential part of eukaryotic gene expression. Although the mechanism of splicing has been extensively studied in vitro, in vivo kinetics for the two-step splicing reaction remain poorly understood. Here, we combine transient transcriptome sequencing (TT-seq) and mathematical modeling to quantify RNA metabolic rates at donor and acceptor splice sites across the human genome. Splicing occurs in the range of minutes and is limited by the speed of RNA polymerase elongation. Splicing kinetics strongly depends on the position and nature of nucleotides flanking splice sites, and on structural interactions between unspliced RNA and small nuclear RNAs in spliceosomal intermediates. Finally, we introduce the ‘yield’ of splicing as the efficiency of converting unspliced to spliced RNA and show that it is highest for mRNAs and independent of splicing kinetics. These results lead to quantitative models describing how splicing rates and yield are encoded in the human genome.Genes are portions of DNA that carry the instructions to build proteins. In particular, they are formed of segments called exons, which contain the protein-building information, and of non-coding segments known as introns. Exons and introns alternate within a gene. To create a given protein, the cell first uses an enzyme, Polymerase II, to copy the entire related gene – including introns and exons – into a molecule of ribonucleic acid, or RNA. As the gene is copied, a machine called the spliceosome comes onto the RNA molecule to remove the introns and create the final RNA template used to produce proteins. The spliceosome works by recognizing specific sequences that signal the border between introns and exons. Once the machine is bound to these ‘splice sites’ on each side of an intron, it brings the two neighboring exons close together and cuts out the intron. The two ends of the exons are then attached together. Previous studies have measured how fast introns are removed, but it remained unclear how long it takes to cut individual splice sites genome-wide. To address this question, Wachutka, Caizzi et al. combined a mathematical approach with a biochemical method that purifies newly made RNA in human cells. The experiments showed that it only took a few minutes to cut most splice sites. Cutting splice sites that bordered very long introns was slower, presumably because the Polymerase II took longer to produce these introns. In addition, the genetic sequences of the splice sites affected the time it took to remove the introns: some made it harder for the spliceosome to recognize where to cut, but others made it easier. Mistakes in removing introns from RNA can lead to producing abnormal proteins, and many diseases such as cystic fibrosis and Duchenne muscular dystrophy can be caused by such errors. In particular, small changes in the sequences at the splice sites or in the surrounding areas can create problems when it comes to eliminating introns. Decrypting the dynamics of intron cutting and removal may give scientists new insight into the molecular causes of cystic fibrosis and many other genetic disorders
Author response
Full text linkTranscription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Oct4 differentially regulates chromatin opening and enhancer transcription in pluripotent stem cells
Full text linkVariations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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