Methods for measuring splicing and transcription rates in living cells

An important step toward understanding gene regulation is the elucidation of the time necessary for the completion of individual steps. Measurement of reaction rates can reveal potential nodes for regulation. For example, measurements of in vivo transcription elongation rates reveal regulation by DNA sequence, gene architecture, and chromatin. Pre-mRNA splicing is regulated by transcription elongation rates and vice versa, yet the rates of RNA processing reactions remain largely elusive. Since the 1980s, numerous model systems and approaches have been used to determine the precise timing of splicing in vivo. Because splicing can be co-transcriptional, the position of Pol II when splicing is detected has been used as a proxy for time by some investigators. In addition to these ‘distance-based’ measurements, ‘time-based’ measurements have been possible through live cell imaging, metabolic labeling of RNA, and gene induction. Yet splicing rates can be convolved by the time it takes for transcription, spliceosome assembly and spliceosome disassembly. The variety of assays and systems used has, perhaps not surprisingly, led to reports of widely differing splicing rates in vivo. Recently, single molecule RNA-seq has indicated that splicing occurs more quickly than previously deduced. On the other hand, introduction of splicing delays through as yet unknown mechanisms provide opportunity for regulation. More work is needed to understand how cells optimize the rates of gene expression for a range of biological conditions.

Comparison of distance-based and time-based measurements of splicing in vivo

rna-seq

Many studies, indicated by flags on the gene ruler (upper panel) or time line (lower panel), have addressed when or where splicing occurs in multiple species and obtained the indicated results. This summary relates the measured half-maximum splicing values (or similar value if half-max not provided). Translation of distance to time is herein assumed at 3 kb/min.

Alpert T, Herzel L, Neugebauer KM. (2016) Perfect timing: splicing and transcription rates in living cells. Wiley Interdiscip Rev RNA [Epub ahead of print]. [article]

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