The Atypical Kinase RIOK3 Limits RVFV Propagation and Is Regulated by Alternative Splicing

Abstract

In recent years, transcriptome profiling studies have identified changes in host splicing patterns caused by viral invasion, yet the functional consequences of the vast majority of these splicing events remain uncharacterized. We recently showed that the host splicing landscape changes during Rift Valley fever virus MP-12 strain (RVFV MP-12) infection of mammalian cells. Of particular interest, we observed that the host mRNA for Rio Kinase 3 (RIOK3) was alternatively spliced during infection. This kinase has been shown to be involved in pattern recognition receptor (PRR) signaling mediated by RIG-I like receptors to produce type-I interferon. Here, we characterize RIOK3 as an important component of the interferon signaling pathway during RVFV infection and demonstrate that RIOK3 mRNA expression is skewed shortly after infection to produce alternatively spliced variants that encode premature termination codons. This splicing event plays a critical role in regulation of the antiviral response. Interestingly, infection with other RNA viruses and transfection with nucleic acid-based RIG-I agonists also stimulated RIOK3 alternative splicing. Finally, we show that specifically stimulating alternative splicing of the RIOK3 transcript using a morpholino oligonucleotide reduced interferon expression. Collectively, these results indicate that RIOK3 is an important component of the mammalian interferon signaling cascade and its splicing is a potent regulatory mechanism capable of fine-tuning the host interferon response.

Keywords: MP-12; RIOK3; Rift Valley fever virus; alternative splicing; innate immune response; morpholino oligonucleotide.

Figure 1

RIOK3 plays an antiviral role during RVFV MP-12 infection. (A) siRNA knockdown of RIOK3. HEK293 cells were transfected with RIOK3 (Qiagen nos. Hs_RIOK3_4 and Hs_RIOK3_8) or control siRNAs. Total RNA was extracted and RIOK3 mRNA was quantified by RT-qPCR. (B) Effect of RIOK3 knockdown on MP-12 titer. HEK293 cells were transfected with mock or RIOK3 siRNAs, then infected with RVFV strain MP-12. The viral titer in the supernatant at 24 hpi was quantified by plaque assay. (C) Effect of RIOK3 knockdown on rLuc RVFV transcriptional activity. HEK293 cells were transfected with siRNA targeted to RIOK3 or a negative control and infected with rLuc RVFV. The Renilla luciferase activity was quantified at 24 hpi and indicated the level of RVFV transcriptional activity. (D) Effect of RIOK3 overexpression on rLuc RVFV transcriptional activity. Cell lysates were prepared from HEK293 cells transfected with N-terminally FLAG tagged RIOK3 or mock transfected and protein levels were determined by Western blotting. Transfected cells were infected with rLuc RVFV and the level of Renilla luciferase activity was quantified at 24 hpi. In all panels, data are presented as mean +/− SEM of duplicate (C) or triplicate (A,B,D) biological replicates. Student’s t-test: *** p < 0.001, ** p < 0.01.

Figure 2

RIOK3 is involved in the signal transduction of the type I IFN response. (A) HEK293 cells and three RIOK3 KO cell lines were screened for RIOK3 protein expression by IP-Western blot. (B) HEK293 cells and RIOK3 KO cells were infected with MP-12 (MOI 1.0). The viral titer in the 24 hpi supernatant was quantified by plaque assay. (C,D) RT-qPCR targeting IFNB was performed on RNA from HEK293 cells and RIOK3 KO cells infected with MP12 (C) or treated with poly (I:C) (D) or 3p-hpRNA (E) for 18 hours. (F) RT-qPCR targeting IFNB was performed on RIOK3 KO cells transfected with a GFP (mock) or RIOK3 expression plasmid and treated with poly (I:C) 18 hours later. (G) Effect of RIOK3 KO on IFNB promoter activation. HEK293 cells and RIOK3 KO cells were co-transfected with pGL3-IFNB firefly reporter and phRL-CMV renilla control. Cells were stimulated with poly (I:C) or infected with MP-12 and the dual luciferase signals were measured after 18 or 48 h, respectively. In panels B–F, data are presented as mean +/− SEM from three biological replicates. Western blots in panels A is representative of duplicate experiments. Student’s t-test: ** p < 0.01, * p < 0.05.

Figure 3

RVFV infection induces alternative splicing of RIOK3 transcripts. Distribution of RIOK3 splicing isoforms in either mock-infected (A) or rLuc RVFV infected (B) HEK293 cells. Total RNA was purified from mock- or virus-infected cells, reverse transcribed/PCR amplified into mRNA-length cDNAs, and cloned in a plasmid. From each sample, 40 individual clones were sequenced and categorized as full length, X1, X2, or X1/X2 hybrid. (C) Prevalence of RIOK3 X2 alternative splicing in infected cells. HEK293 cells were infected (MOI 1.0) with either MP-12 or rLuc RVFV and total RNA was extracted after 24 h. The fraction of X2 variant alternative splicing was quantified by RT-qPCR using primers specific to detect the canonical and X2 isoforms at exons 8 and 9, and not necessarily in full length poly-adenylylated mRNAs, which were characterized in panels (A,B). Data in panel C is presented as mean +/− SEM from triplicate experiments. (D) Schematic of the different splicing patterns observed. X2 employs a cryptic splice donor site within exon 8, resulting in a shortened exon 8 and a new stop codon in exon 9. X1 skips exon 7 entirely. Some transcripts contained both X1 and X2 type alternative splicing.

Figure 4

Activation of cytosolic innate immune RNA sensors, but not DNA sensors induces RIOK3 X2 variant alternative splicing, and RIOK3 splicing is vital for IFN expression. HEK293 cells were transfected with poly (I:C) (A), or infected with either the RNA virus Tacaribe (TCRV) (B), or the DNA virus adenovirus (ADV) (C). Total RNA was harvested 24 h post transcription or infection and RT-qPCR was used to quantify the relative fraction of X2 variant and full-length canonically spliced RIOK3 species. Data are presented as mean +/− SEM based on duplicate experiments. Student’s t-test: * p < 0.05. (D) HEK293 cells were transfected with 1 μg/mL 3p-hpRNA. RT-PCR targeted the region spanning RIOK3 exons 5–10, followed by agarose gel electrophoresis. Splicing isoforms are indicated. (E) Morpholino oligonucleotide targeting the canonical exon 8 splice donor site of RIOK3 pre-mRNA was transfected into HEK293 cells in increasing concentration (2–10 μM) for 18 h. RNA was processed via RT-PCR and run on agarose gel. (F) RT-qPCR was performed to measure the expression of IFNB mRNA after 18 h MO (8 μM) transfection and subsequent stimulation by either poly (I:C) or 3p-hpRNA. Data is presented as mean +/− SEM from triplicate experiments. Student’s t-test: ** p < 0.01, * p < 0.05.

Figure 5

RIOK3 X2 variant mRNA encodes a truncated protein product and is targeted by nonsense-mediated decay (NMD). (A) Schematic of the normal and X2 variant alternative splicing on the precursor RIOK3 RNA transcript together with a representation of the full-length and X2 variant RIOK3 protein isoforms. (B) Alternatively spliced RIOK3 mRNA is efficiently degraded by nonsense mediated decay. HEK293 cells were mock treated or treated with cycloheximide (CHX) for the times indicated. Total RNA was harvested and subjected to RT-PCR using primers flanking the spliced exons. PCR products were run on agarose and stained using ethidium bromide. As CHX inhibits NMD, X2 and X1/X2 products are preserved with treatment. Actin B was used as a control. (C) Expression of RIOK3 full-length and X2 variant proteins from transfected expression plasmids. RIOK3 full-length and X2 constructs were transfected into HEK293 cells and expression was analyzed by Western blot. The presence of the same FLAG tag at the N-terminus of both proteins allowed us to compare their expression efficiencies.

Figure 6

Relationship between RIOK3 splicing products, RVFV MP-12 infection, and innate immune activation. The human RIOK3 gene is represented at the top of the panel with its 13 exons. The normal RIOK3 protein with a complete kinase domain is called full-length and produced by translation of a canonically spliced transcript encompassing the 13 exons (left side of figure). Alternative 5′ splice donor usage shortens exon 8 and results in the X2 variant mRNA. Translation of the X2 mRNA would produce a truncated RIOK3 missing most of the kinase domain (right side of figure). Notably, the X2 RNA contains an exonic premature termination codon making it a canonical substrate for nonsense-mediated decay and rapid degradation. The effects on RIOK3 gene expression during MP-12 infection (blue arrows) compared to uninfected cells (orange arrows) are shown. The magnitude of the effect is represented by the thickness of each arrow. It is not known if the X2 variant RIOK3 protein is expressed or stable, or if it possesses any function. By contrast, the full-length RIOK3 protein has been suggested to be involved in several diverse cell functions and pathways (see text), thus the relative expression levels of full length and X2 variants of RIOK3 can have strong effects on cellular functions including the antiviral response.