Perfect timing: splicing and transcription rates in living cells

Abstract

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. Here we comprehensively review these findings and discuss evidence that splicing and transcription rates are closely coordinated, facilitating the efficiency of gene expression. 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. WIREs RNA 2017, 8:e1401. doi: 10.1002/wrna.1401 For further resources related to this article, please visit the WIREs website.

Figure 1. Observation of co-transcriptionally spliced transcripts leads to distance-based view of splicing kinetics in vivo

A) Electron micrograph of chromatin spreads from Drosophila melanogaster (left panel) visualize electron dense spliceosomes as they assemble near the 5’ ends of nascent transcripts; shortening of transcript 10 is indicative of intron removal (77). Camera lucida drawing of the chromatin spread is shown in the right panel. B) Diagram of a simple gene with a single intron undergoing transcription by several active Pol II molecules. The 5’ methyl cap (black ball) is added shortly after transcription of the 5’ end of the RNA transcript. Spliceosomal components (red balls) bind the 5’SS and 3’SS of the pre-mRNA co-transcriptionally.

Figure 2. Comparison of distance-based and time-based measurements of splicing in vivo

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 (78). See text for details.

Figure 3. Time-based experiments vary widely in methodology and results

A) The median number of exon-intron junctions are processed in 14 min according to RNA-seq reads of metabolically labeled mouse RNA (44). B) Use of fluorescent reporter genes permits imaging of introns and quantitation of their half-lives (50). C). Fraction spliced values from a metabolic labeling experiment are plotted at different time points for three pairs of gene paralogs in yeast. Paralogs have identical exonic sequences and different intronic sequences. Splicing values appear highly similar between paralogs, yet different between genes (57).

Figure 4. Recent distance-based techniques predict spliceosome adjacent to polymerase during splicing

A) Representative SMIT trace from budding yeast shows splicing reaches half-maximum levels at ~62 bp past the 3’ SS (59). B) Long read sequencing of budding yeast genes enables splicing analysis of single molecules (59). C) 3D reconstruction of nascent transcription and splicing complex (NTS) bound to Balbiani ring 3 locus on chromatin (dashed line) from Chironomus tentans with antibody gold particles against U2 snRNP (left) and Pol II CTD (right) (60). D) Crystal structures are docked into electron microscopy density show that capping enzymes bind RNA exit tunnel of RNA Polymerase II to modify the 5’ end of RNA immediately (5).