DDX1 is required for non-spliceosomal splicing of tRNAs but not of XBP1 mRNA

Abstract

RNA helicase DEAD-box helicase 1 (DDX1) forms a complex with the RNA ligase 2´,3´-cyclic phosphate and 5´-OH ligase (RTCB), which plays a vital role in non-spliceosomal splicing of tRNA and X-box binding protein 1 (XBP1) mRNA. However, the importance of DDX1 in non-spliceosomal splicing has not been clarified. To analyze the functions of DDX1 in mammalian cells, we generated DDX1 cKO cells from the polyploid human U2OS cell line and found that splicing of intron-containing tRNAs was significantly disturbed in DDX1-deficient cells, whereas endoplasmic reticulum (ER) stress-induced splicing of XBP1 mRNA was unaffected. Additionally, the enforced expression of DDX1, but not of its helicase-inactive mutant, rescued the splicing defects of tRNAs in DDX1-deficient cells. These results indicate that RTCB is required for the splicing of both tRNA and XBP1 mRNA, whereas the DDX1 enzymatic activity is specifically required for tRNA splicing in vivo.

Fig. 1. Establishment of DDX1 cKO U2OS cells via all-in-one cKO method.

A Strategy to generate U2OS DDX1 cKO cells. An arrow indicates the CRISPR/Cas9 target site. White triangle, FRT sequence; arrowhead, primers for genome PCR; N, NdeI recognition sequence. The blue box represents exon 3 upstream of the CRISPR/Cas9 cleavage site. The red box represents exon 3 downstream of the CRISPR/Cas9 cleavage site. The yellow box represents the ORF of exon 2. B Twelve isolated clones were analyzed by genome PCR. The upper band represents the targeted allele, while the lower band represents the non-targeted allele. The non-targeted band of clone 7 is larger than that of control, which is likely due to a large insertional mutation in the non-targeted allele caused by the function of CRISPR/Cas9. C Expression of DDX1 in the indicated clones was analyzed via western blotting. C, U2OS cells. D NdeI-digested genomic DNA of clones #2, #8, and #10 was analyzed by Southern blotting. C, U2OS cells. Clone #2 was used as U2OS DDX1 cKO cells hereafter. E The expression of DDX1 was analyzed by qRT-PCR (n = 3; NS, not significant). F The expression of the fluorescent protein was analyzed via flow cytometry. Cells were analyzed on day 2 after sorting. G Expression levels of DDX1 in EGFP-positive (cKO^F) and EGFP-negative (KO) cells were analyzed via western blotting. H The number of DDX1 cKO^F and KO cells were counted on the indicated days after sorting (n = 3; *P < 0.05; ***P < 0.005).

Fig. 2. Function of DDX1 in non-spliceosomal splicing.

A Splicing pathway of intronic tRNAs in humans. TSEN cleaves off intron from pre-tRNA, and RTCB ligates the 5´ and 3´ exon of tRNAs. B Total RNA (2.5 µg) was loaded to analyze splicing of indicated tRNAs by northern blotting with oligo-probes designed on 5´ exon of tRNAs. The samples were harvested on day 8 after sorting KO cells. Black triangle, mature tRNA; white triangle, intermediate tRNA. C The ratio of intermediate and mature tRNA was quantified (n = 3; *P < 0.05; **P < 0.01; ***P < 0.005). D Expression levels of DDX1-binding factors in the DDX1 cKO^F and KO cells were analyzed via western blotting. E Schematic representation of the primer position to detect XBP1u and XBP1s expression via endpoint RT-PCR and qRT-PCR. Blue arrowheads, primers for endpoint RT-PCR; black arrowhead, a primer for qRT-PCR of XBP1u; white arrowhead, a primer for qRT-PCR of XBP1s; gray arrowheads, a qRT-PCR primer common to XBP1s and XBP1u. F DDX1 cKO^F, KO, and ERN1 KO cells were treated with 10 µg/ml tunicamycin for the indicated period, and the expression levels of XBP1 and XBP1u were detected via endpoint RT-PCR. (G) DDX1 cKO^F, KO, and ERN1 KO cells were treated with 10 µg/ml tunicamycin for the indicated period, and the expression of XBP1s was analyzed via qRT-PCR (n = 3; NS, not significant; ***P < 0.005). H The ratio of XBP1s to XBP1u mRNA expression was calculated based on qRT-PCR data (n = 3; NS, not significant; *P < 0.05). I DDX1 cKO^F, KO, and ERN1 KO cells were treated with 10 µg/ml tunicamycin for the indicated period, and the expression of ERDJ4 was analyzed via qRT-PCR (n = 3; NS, not significant; *P < 0.05).

Fig. 3. RTCB activity is required for both tRNA and XBP1 mRNA splicing.

A Total RNA (2.5 µg) was loaded to analyze splicing of Tyr-GTA tRNAs in RTCB WT and KO cells by northern blotting with an oligo-probe designed on 5´ exon of Tyr-GTA tRNAs. The samples were harvested on day 3 after sorting transfected cells. Black triangle, mature tRNA; white triangle, intermediate tRNA. B The ratio of intermediate and mature tRNA was quantified (n = 3; *P < 0.05). C WT, RTCB KO, and Archease KO cells were treated with 10 µg/ml tunicamycin for the indicated period, and the expression of XBP1s was analyzed via qRT-PCR (n = 3; ***P < 0.005). D The ratio of XBP1s to XBP1u mRNA expression was calculated based on qRT-PCR data (n = 3; NS, not significant; **P < 0.01; ***P < 0.005). E RTCB WT and KO cells were treated with 10 µg/ml tunicamycin for the indicated period, and the expression of ERDJ4 was analyzed via qRT-PCR (n = 3; NS, not significant; **P < 0.01). F Total RNA (2.5 µg) was loaded to analyze Splicing of Tyr-GTA tRNAs in Archease WT and KO cells by northern blotting with an oligo-probe designed on 5´ exon of Tyr-GTA tRNAs. The samples were harvested on day 3 after sorting transfected cells. Black triangle, mature tRNA; white triangle, intermediate tRNA. G The intermediate to mature tRNA ratio was quantified (n = 3; *P < 0.05). H Archease WT and KO cells were treated with 10 µg/ml tunicamycin for the indicated period, and the expression of ERDJ4 was analyzed via qRT-PCR (n = 3; NS, not significant; ***P < 0.005).

Fig. 4. Enzymatic activity of DDX1 is required for tRNA splicing.

A Expression of exogenously expressed DDX1 in the DDX1 KO cells was analyzed by western blotting. B Infection efficiency was verified with the expression of Ruby, a red fluorescent protein, linked to the introduced gene of interest via the P2A peptide sequence. Red, DDX1 KO U2OS cells; gray, lentiviral vector infected DDX1 KO U2OS cells. C Total RNA (2.5 µg) was loaded to analyze splicing of Tyr-GTA tRNA in DDX1 wt or K52N mutant introduced DDX1 KO U2OS cells by northern blotting with an oligo-probe designed on 5´ exon of Tyr-GTA tRNAs. Black triangle, mature tRNA; white triangle, intermediate tRNA. D The ratio of intermediate and mature tRNA was quantified (n = 3; NS, not significant; ***P < 0.005).

Fig. 5. Proposed model of DDX1 function in tRNA splicing.

DDX1 could be specifically required for remodeling the intermediate tRNA structure for the RTCB-catalyzed ligation reaction. Filled circles and a white circle represent anticodon sequence and 3´ terminus of 5´ exon of pre-tRNA, respectively.