Splicing kinetics and transcript release from the chromatin compartment limit the rate of Lipid A-induced gene expression

Abstract

The expression of eukaryotic mRNAs is achieved though an intricate series of molecular processes that provide many steps for regulating the production of a final gene product. However, the relationships between individual steps in mRNA biosynthesis and the rates at which they occur are poorly understood. By applying RNA-seq to chromatin-associated and soluble nucleoplasmic fractions of RNA from Lipid A-stimulated macrophages, we examined the timing of exon ligation and transcript release from chromatin relative to the induction of transcription. We find that for a subset of genes in the Lipid A response, the ligation of certain exon pairs is delayed relative to the synthesis of the complete transcript. In contrast, 3′ end cleavage and polyadenylation occur rapidly once transcription extends through the cleavage site. Our data indicate that these transcripts with delayed splicing are not released from the chromatin fraction until all the introns have been excised. These unusual kinetics result in a chromatin-associated pool of completely transcribed and 3 ′ -processed transcripts that are not yet fully spliced. We also find that long introns containing repressed exons that will be excluded from the final mRNA are excised particularly slowly relative to other introns in a transcript. These results indicate that the kinetics of splicing and transcript release contribute to the timing of expression for multiple genes of the inflammatory response.

Induced transcription and stability of CELF2 mRNA drives widespread alternative splicing during T-cell signaling

“…Despite this, we demonstrate here that Dicer-mediated microRNA mechanisms play a minimal role in regulating CELF2 mRNA in developing thymocytes. Alternative mechanisms for regulating mRNA stability include NMD and nuclear mRNA retention, both of which have also been implicated as important to various aspects of immune cell physiology (32,46,47). However, we also show here that neither NMD nor nuclear retention play a significant role in the stability or expression of any of the CELF2 isoforms.…”

“…In a second potential mechanism, intron 13 retention could lead to nuclear retention of the resulting mRNA, which in turn is sometimes manifest in transcript accumulation (30)(31)(32). To address the possibility that intron retention in the 3′UTR alters the nucleo-cytoplasmic localization of CELF2 mRNA, we fractionated cells and assayed for RNA abundance in the nuclear and cytoplasmic pools (Fig.…”

Detained introns are a novel, widespread class of post-transcriptionally spliced introns

“…The latter may be related to a recently discovered developmental regulation mechanism in which inhibition of splicing of a 39-terminal intron leads to destruction of the transcript through nuclear degradation (Yap et al 2012). The possibility that many DIs represent a rate-limiting intermediate is suggestive of a process observed in recent studies using lipid A-induced macrophages (Bhatt et al 2012;Pandya-Jones et al 2013) in which there is a delay of tens of minutes between transcription of a gene and the removal of all introns, which occurs in a chromatin-associated fraction, thus demonstrating that splicing rates can determine the kinetics of gene expression. However, in these cases, all or most introns in the genes appeared to be similarly delayed, whereas DIs are distinctly present in transcripts that are otherwise fully spliced.…”

“…This observation is unexpected in light of several recent transcriptome-wide analyses suggesting that completion of most splicing events occurs cotranscriptionally, prior to transcription termination and polyadenylation (Carrillo Oesterreich et al 2010;Ameur et al 2011;Khodor et al 2011;Tilgner et al 2012). On the other hand, there is extensive literature proposing post-transcriptional splicing of individual introns (Bauren and Wieslander 1994;Smith et al 1999;Melcak et al 2001;Vargas et al 2011;Hao and Baltimore 2013;Pandya-Jones et al 2013). In addition, other RNA-seq-based studies examining biochemically fractionated cells, metabolically labeled RNA, or lipid A-induced transcriptional activation and tracking the maturation of nascent transcripts from chromatin to cytoplasm have indicated varying degrees of posttranscriptional splicing (Rabani et al 2011;Bhatt et al 2012;Khodor et al 2012;Windhager et al 2012;PandyaJones et al 2013).…”

“…More recently, high-throughput sequencing studies in multiple organisms have detected the presence of introns in poly(A) + or fully transcribed, chromatin-associated RNA populations, indicating that some fraction of splicing occurs post-transcriptionally (Ameur et al 2011;Khodor et al 2011Khodor et al , 2012Rabani et al 2011;Bhatt et al 2012;Tilgner et al 2012;Windhager et al 2012;Pandya-Jones et al 2013;Shalgi et al 2014;Braunschweig et al 2014). Prior studies have observed that the kinetics of removal of alternatively spliced introns are slower than those of the typical constitutive intron (Pandya-Jones and Black 2009; Khodor et al 2011;Vargas et al 2011;Pandya-Jones et al 2013). Here, it has been suggested that the kinetics may be intrinsic to the splicing outcome; i.e., that constitutive splicing is accomplished cotranscriptionally, while regulated variants are spliced more slowly.…”