Balance between MAT2A intron detention and splicing is determined cotranscriptionally

Abstract

Transcriptome analysis of human cells has revealed that intron retention controls the expression of a large number of genes with diverse cellular functions. Detained introns (DI) constitute a subgroup of transcripts with retained introns that are not exported to the cytoplasm but instead remain in the nucleus. Previous studies reported that the splicing of DIs in the CLK1 transcript is post-transcriptionally induced to produce mature mRNA in the absence of new transcription. Thus, CLK1-DI serves as a precursor or "reservoir" for the CLK1 mRNA. However, whether this is a universal mechanism for gene regulation by intron detention remains unknown. The MAT2A gene encodes S-adenosylmethionine (SAM) synthetase and it contains a DI that is regulated in response to intracellular SAM levels. We used three independent assays to assess the precursor-product relationship between MAT2A-DI and MAT2A mRNA. In contrast to CLK1-DI, these data support a model in which the MAT2A-DI transcript is not a precursor to mRNA but is instead a "dead-end" RNA fated for nuclear decay. Additionally, we show that in SAM-deprived conditions the cotranscriptional splicing of MAT2A detained introns increases. We conclude that polyadenylated RNAs with DIs can have at least two distinct fates. They can serve as nuclear reservoirs of pre-mRNAs available for rapid induction by the cell, or they constitute dead-end RNAs that are degraded in the nucleus.

Keywords: MAT2A; S-adenosylmethionine; intron detention; intron retention; splicing.

FIGURE 1.

Assessing precursor–product relationship between CLK1-DI, MAT2A-DI, OGT-DI and their cognate mRNAs by transcription inhibition. (A) Diagrams of the reservoir and dead-end models for induction of detained introns. In the reservoir model, poor cotranscriptional splicing generates nuclear RNAs with a specific intron detained. These RNAs can then be post-transcriptionally induced for rapid mRNA production independent of new transcription. In contrast, the dead-end model proposes that the splicing choice occurs cotranscriptionally. In uninduced states, the intron is largely detained and the DI RNA is degraded. Upon induction, cotranscriptional splicing efficiency increases. See text for additional details. (B) Northern blots demonstrating CLK1 (left) and MAT2A (right) isoform switching upon CB19 treatment and Met depletion, respectively. In all northern blots, the detained intron and mRNAs isoforms are labeled “DI” or “m,” respectively. The asterisk in the CLK1 northern represents cross-hybridization to the rRNA and serves as a loading control. (C) Experimental timeline (top) and representative northern blot for the CLK1 splicing induction and transcription inhibition assay. (D) Quantification of the CLK1 induction experiment. CLK1 signals were first normalized to GAPDH to control for loading in each lane. GAPDH does not appreciably degrade over the time course in this experiment. The value for CLK1-DI was set to one for each biological replicate. Data represented as mean ± standard deviation (SD) (n = 3); asterisk represents P < 0.05 in an unpaired, two-tailed Student's t-test. (E) Experimental timeline (top) and representative northern blot for the MAT2A splicing induction and transcription inhibition assay. In the diagrams on the right, the lower case “a”s represent hyperadenylated poly(A) tails. (F) Quantification of the MAT2A induction experiment as in panel D. The spliced nuclear hyperadenylated form was excluded from the analysis. Data represented as mean ± SD (n = 3). (G) Representative northern blot showing the induction of OGT splicing upon OSMI-1 treatment for the indicated times. Here, we quantified the OGT data as percent DI [DI/(DI + mRNA) × 100], to control for high variability in OGT levels of both isoforms between samples. (H) Experimental timeline (top) and representative northern blot for the OGT splicing induction and transcription inhibition assay. The bar graph below is quantification of these northern blot data; data represented as mean ± SD (n = 4).

FIGURE 2.

Assessing precursor–product relationship between MAT2A-DI and MAT2A mRNAs by 4SU pulse-chase. (A) Timeline for the 4SU experiment. Note that the T = 0 samples are those that are harvested after the 1-h washout period. (B) Representative northern blot of a 4SU pulse-chase experiment. The “input” samples are total RNA while the 4SU samples have been selected for 4SU incorporation. The “No 4SU” samples were collected at T = −3 h. (C) Quantification of the northern blot data. After normalizing to GAPDH (or rRNA) to control for loading and subtraction of the “No 4SU” signal as background, the additive value of the mRNA and DI isoforms for the T = 0 samples was set to one. The quantity of each isoform for each time point was then referenced to this value to normalize between experiments. As a result, these graphs provide information on both the relative levels and ratios of DI and mRNA isoforms. Data represented as mean ± SD (n = 3). D and E are the same as panels B and C, except that a cDNA transgene of MAT2A was overexpressed in the cells (Pendleton et al. 2017) (n = 4).

FIGURE 3.

Inhibition of PPD does not increase MAT2A mRNA levels after induction. (A) Cells were treated with nontargeting (siCtrl) or PAPα/γ (siPAP) siRNAs and were subject to Met depletion as indicated. Representative northern blots (top) and quantification of the data (bottom) are shown. All values were normalized to GAPDH as a loading control, then the untreated mRNA value was set to one for each sample. Data represented as mean ± SD (n = 3). B and C are similar experiments, except PPD was inhibited by LALA overexpression or cordycepin treatment, respectively.

FIGURE 4.

MAT2A and CLK1 splicing is cotranscriptionally enhanced upon induction. (A) Diagram of MAT2A gene with the positions of the quantitative RT-PCR primer pairs shown; diagram is not to scale. The splicing of exons 1, 2, and 3 is constitutive while 8–9 flank the DI. For all splice junction RNAs (e.g., amplicons A and B), the unspliced product is too large to amplify under our qPCR conditions. (B) Experimental scheme (top) and analysis of RNA from a NRO experiment. The RT-qPCR values were first normalized to GAPDH signal and then referenced to the +Met control for the same amplicon, which was set to one. No 4SU controls showed negligible signal. Data represented as mean ± SD (n ≥ 5); the asterisk represents P < 0.05 in an unpaired, two-tailed Student's t-test. (C) Experimental scheme (top) and analysis (bottom) of RNA from a 4SU quick-pulse experiment. The normalization and quantification of RT-qPCR data were performed as in panel B (n ≥ 4). (D) Cells overexpressing flag-tagged wild-type or mutant METTL16 were subject to a quick-pulse assay as in panel C. All cells were grown in Met-replete media. All three proteins are expressed at similar levels under these conditions (Pendleton et al. 2017). P-values were referenced to vector control (n = 3). The vector alone control samples were set to one. (E) Quick-pulse analysis of the CLK1 RNA with primers indicated on the diagram (top). Analysis was performed as described for panel C; in this case, DMSO was the reference sample for each primer set (n = 3).