CLK-dependent exon recognition and conjoined gene formation revealed with a novel small molecule inhibitor

Abstract

CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3′-end processing and associated splicing factors.

Anti‐tumor efficacy of a novel CLK inhibitor via targeting RNA splicing and MYC‐dependent vulnerability

“…Interestingly, our RNA-Seq data suggested that cellular status of MYC slightly affected the determination of downstream AS events in response to CLK inhibition because T-025 caused similar AS in four tested solid cancer cell lines, including MYC-amplified COLO320HSR cells. The finding that MYC slightly affected CLK inhibitor-dependent AS events was also supported by a previous finding that CLK inhibitor, T3-dependent AS events mostly (~75%) overlapped between the hTERT un-transformed fibroblast cells and MYC-amplified HCT116 colorectal cancer cells (Funnell et al, 2017). We also show that transcriptional regulation of CLK2 and other CLK kinases were independent on MYC activation, degree of T-025-mediated SE was similar regardless MYC induction, and AS events regulated by MYC induction or T-025 rarely overlapped.…”

“…of three independent experiments ( n = 3). IC 50 values and 95% CI were determined by using Prism. Data information: The chemical structure of T3, the result of enzymatic assay of T3, and AS events modulated by T3 in HCT116 are cited from a previous article (Funnell et al , ).…”

“…First, the different chemical scaffold CLK inhibitor T3 exhibited similar effects to T‐025 in terms of AS and the profile of growth inhibition, including MYC induction‐dependent higher activity. Notably, AS caused by T3 has been reported to be largely overlapped with that caused by CLK1/2/3/4 siRNA (Funnell et al , ), suggesting that AS caused by T‐025 was also similar to that caused by the depletion of CLKs. Second, we show that expression level of CLK2 is responsible for the T‐025‐mediated effects on the induction of AS and the suppression of cell proliferation; The SE type AS caused by T‐025 was larger in CLK2 high expressing NCI‐H1048 cells, and smaller in CLK2 low expressing 786‐O cells.…”

“…2018 Takeda Pharmaceutical Company EMBO Molecular Medicine 10: e8289 | 2018effects to T-025 in terms of AS and the profile of growth inhibition, including MYC induction-dependent higher activity. Notably, AS caused by T3 has been reported to be largely overlapped with that caused by CLK1/2/3/4 siRNA(Funnell et al, 2017), suggesting that AS caused by T-025 was also similar to that caused by the depletion of CLKs. Second, we show that expression level of CLK2 is responsible for the T-025-mediated effects on the induction of AS and the suppression of cell proliferation; The SE type AS caused by T-025 MYC activation and T-025 treatment synergistically induced apoptosis.A, B Dox treatment-induced/activated MYC in MYC-inducible SK-MEL-28 cells.…”

Functional diversification of Ser-Arg rich protein kinases to control ubiquitin-dependent neurodevelopmental signalling

“…However, SRPK family members exhibit highly tissue-specific expression profiles (Nakagawa et al, 2005; Wang et al, SRPKIN-1 (Hatcher et al, 2018) (Figure 1A). In contrast, treatment of mESCs with T3, a selective inhibitor of the closely related CLK kinases (Funnell et al, 2017), which have also been shown to phosphorylate splicing factors (Colwill et al, 1996), leads to near complete inhibition of SR-protein phosphorylation ( Figure 1A). Our results suggest that SRPKs are not the major SRSF protein kinases in mESCs, and may have acquired other developmental function(s) during metazoan evolution.…”