Figures and data in tRNA ligase structure reveals kinetic competition between non-conventional mRNA splicing and mRNA decay

Figures
Tables
Additional files

6 figures, 2 tables and 4 additional files

Figures

Figure 1 with 1 supplement

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Fungal tRNA ligase Trl1.

(A) Domain organization of Trl1. The ligase/adenylyltransferase domain (LIG) is shown in blue, the polynucleotide kinase domain (KIN) in white, the cyclic phosphodiesterase domain (CPD) in green. The residue numbering refers to the domain boundaries of Trl1 from *Saccharomyces cerevisiae* (Sc) and *Chaetomium thermophilum* (Ct). The relative position of the mutated histidine in the UPR mutant is indicated. See Figure 1—figure supplement 1 for a sequence alignment of Trl1-LIG from both species. (B) Trl1-mediated non-conventional RNA splicing mechanism. The RNA is cleaved at the exon-intron junctions by an endonuclease (SEN or Ire1), which leaves a 2’,3’-cyclic phosphate end on the 5’ exon (purple) and a 5’-OH on the 3’ exon (yellow). After removal of the intron (gray), the exon ends are first modified (‘healed’) by the CPD and KIN domains and finally ligated (‘sealed’) by the LIG domain. Exon ligation progresses via a covalent ligase-AMP intermediate (LIG-AMP) and a 5’−5’ RNA-adenylate. The residual 2’-phosphate at the splice site is then removed by a separate enzyme, the 2’-phosphotransferase Tpt1.

https://doi.org/10.7554/eLife.44199.002

Figure 1—figure supplement 1

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Sequence alignment between scTrl1 and ctTrl1.

Sequence alignment of Trl1-LIG from *Chaetomium thermophilum* (ct) and *Saccharomyces cerevisiae* (sc). White letters with gray or black background indicate similarity or identity, respectively. The active site lysine, which is part of the highly conserved motif I, and the histidine that is mutated to tyrosine in the UPR mutant are highlighted in green and red, respectively.

https://doi.org/10.7554/eLife.44199.003

Figure 2 with 1 supplement

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Functional complementation by thermophilic ctTrl1.

(A) Functional complementation in *S. cerevisiae trl1Δ* by ctTrl1. Expression of ctTrl1 from a plasmid (*URA3* selection) permitted growth of a *trl1Δ* strain under normal growth conditions (SC-URA) and ER stress (SC-URA +Tm). Counterselection against the *URA3*-containing plasmid using 5’-fluoroorotic acid (5-FOA) confirmed the essential function of Trl1. The respective *TRL1* genotype and plasmid are indicated in the key on top. (B) In vitro splicing of a *HAC1*-derrived RNA substrate was analyzed by denaturing urea-PAGE. The input RNA (lane 1) was first cleaved by scIre1 yielding three dominant bands for the separated exons and the intron (lane 2). Faint bands corresponding to single cleaved RNA were observed. Addition of ctTrl1 completed the splicing reaction resulting in covalently ligated exons (lane 3, marked by a closed arrowhead). An additional ligation product is marked with an asterisk. RNase H digestion identified the band as circularized tandem *HAC1* intron RNA (see Figure 2—figure supplement 1). The position on the gel and relative length of the RNA species is depicted by icons on the right using the same color code as in Figure 1B.

https://doi.org/10.7554/eLife.44199.004

Figure 2—figure supplement 1

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RNase H assay.

In vitro transcribed *HAC1*^U-508 RNA was cleaved by Ire1 (lane 1) and subsequently ligated by Trl1 (lane 2). The ligation products were hybridized with DNA oligonucleotides a (lane 4) or b (lane 5) and treated with RNase H. In lane 3, no oligonucleotide was added. The RNA products after each step were analyzed on a denaturing polyacrylamide gel and visualized by SYBR Gold staining. The band in question (marked with an asterisk) disappeared upon RNase H digestion of the RNA/DNA hybrids and a band at the position of the severed intron (252 nt) reappeared. Based on this result and the position on the gel, the marked band represents most likely two introns that were circularized by two intermolecular ligation events at their 5’ and 3’ ends. Hybridization positions of the DNA oligonucleotides on a linear *HAC1* intron and the putative circular concatemer of two introns are schematically described in the right.

https://doi.org/10.7554/eLife.44199.005

Figure 3

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Structure of ctTrl1-LIG.

(A) Cartoon representation of the overall structure of ctTrl1-LIG from three views. The adenylyltransferase domain is colored in blue and the C-terminal domain in yellow. AMPcPP and the sulfate ion near the active site are depicted as sticks. (B) Electrostatic surface potential map of ctTrl1-LIG. Positive potential is represented in blue and negative potential in red. (C) ATP binding pocket with bound AMPcPP. The interacting amino acid residues and AMPcPP are shown as sticks. Atomic contacts between ctTrl1-LIG and AMPcPP are indicated by dashed lines, with yellow for hydrogen bonds, orange for π-stacking and green for salt bridges. The electron density around AMPcPP (blue mesh) is extracted from a composite omit map (2mF_o-DF_c), contoured at 1 σ. (D) Sulfate ion binding near the active site. Amino acids, AMPcPP and the sulfate ion are shown as sticks. C atoms are colored according to the respective domains (see A) in blue (adenylyltransferase domain) or yellow (C terminal domain). Atomic contacts are depicted as in C.

https://doi.org/10.7554/eLife.44199.006

Figure 4 with 1 supplement

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The UPR mutant *trl1-H148Y* compromises RNA ligation kinetics of Trl1.

(A) Functionally important residues of Trl1-LIG. The structure of ctTrl1-LIG is depicted as surface model, AMPcPP and the sulfate ion are shown as sticks. The active site Lys is depicted in green, His182 (equivalent to His148 in *S. cerevisiae*) in red and residues that have been previously identified as essential by mutagenesis in pink (Wang and Shuman, 2005). (B) In vitro splicing of a *HAC1*-derrived RNA substrate was analyzed by denaturing urea-PAGE. The input RNA (lane 1) was first cleaved by scIre1 yielding three dominant bands for the separated exons and the intron (lane 2). Ligation efficiency was compared by titrating purified scTrl1-LIG-WT (lanes 3–6) or scTrl1-LIG-H148Y (lanes 7–10) to the splicing reaction. Full-length ctTrl1 was used as positive control (lane 11). An additional ligation product is marked with an asterisk. RNase H digestion identified the band as circularized *HAC1* intron RNA (see Figure 2—figure supplement 1). (C) Time-course denaturing urea-PAGE of RNA oligonucleotide ligation by WT or H148Y mutant scTrl1-LIG. The 5’ fluorescein (FAM)-labeled 10 nt *HAC1* 5’ exon oligonucleotide (depicted in orange, lower band) and its ligation product (upper band) with a 20 nt *HAC1* 3’ exon oligonucleotide (depicted in gray) was monitored using fluorescence detection. A representative gel out of three replicates is shown. (D) Quantification of the RNA ligation assay in C. The relative amounts of ligated product in the presence of scTrl1-LIG-WT (black squares) or scTrl1-LIG-H148Y (red circles) were fitted to a first-order model to determine the depicted half-lives. Values represent mean and standard deviation of technical replicates (n = 3).

https://doi.org/10.7554/eLife.44199.008

Figure 4—figure supplement 1

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RNA binding assay.

Cy5-labeled RNA oligonucleotides derived from the *HAC1* sequences of the 5’ and 3’ exon splice sites were incubated with a dilution series of scTrl1-LIG-WT or -H148Y. The concentration-dependent changes in Cy5 fluorescence were fitted to a Hill model of binding. The calculated apparent dissociation constants (K_D) are depicted next to the respective ligase/RNA combination.

https://doi.org/10.7554/eLife.44199.009

Figure 5 with 1 supplement

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*HAC1* mRNA splicing competes with general RNA decay pathways.

(A) Overexpression of various Trl1 constructs rescued the growth phenotype of *trl1-H148Y*. The constructs from *S. cerevisiae* harbored the H148Y mutation of the *trl1-H148Y* allele, whereas those from *C. thermophilum* harbored the equivalent H182Y mutation. Both the full-length proteins and the isolated ligase domains restored growth of the UPR mutant strain when streaked on tunicamycin-containing YPD plates. (B) Serial five-fold dilutions of wild-type and *trl1-H148Y* cells with wild-type or abrogated (*ski2Δ* or *xrn1Δ*) RNA decay pathways were spotted on YPD plates without (-Tm) or with (+Tm) 0.1 μg/ml tunicamycin and incubated at 30°C for 2 days. (C) Model of *HAC1* mRNA splicing and the impact of RNA decay pathways on degradation of cleaved *HAC1* exons. Ire1 cleaves unspliced *HAC1* mRNA (*HAC1*^U) at the non-conventional splice sites upon ER stress. In wild-type yeast cells (*TRL1*), exon-exon ligation by Trl1 is the predominant reaction yielding spliced *HAC1* mRNA (*HAC1*^S). In the context of a kinetically compromised tRNA ligase (*trl1-H148Y*), the cleaved *HAC1* exons are degraded by exonucleolytic RNA decay pathways. The capped (m⁷G) 5’ exon is susceptible to degradation in 3’-to-5’ direction by the RNA exosome (exo)/Ski complex (Ski); the 3’ exon is degraded from its 5’ end by Xrn1. (D) RT-PCR analysis of *HAC1* mRNA splicing in the same strains as in B without and with tunicamycin (Tm). The top panel shows priming outside the exon-intron junctions to distinguish unspliced (U) from spliced (S) *HAC1* mRNA. The relative amount of *HAC1*^S (in %) is indicated below each lane. Both middle panels show the results for priming of the 5’ exon. 18S rRNA was used as a control (bottom panel). The method of cDNA production is indicated on the right of each panel.

https://doi.org/10.7554/eLife.44199.010

Figure 5—figure supplement 1

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The rescue of UPR-deficiency in *trl1-H148Y* is dependent on *HAC1*.

The indicated strains (key in the left) were tested for growth on YPD plates without (left plate) and with (right plate) tunicamycin (Tm).

https://doi.org/10.7554/eLife.44199.011

Figure 6

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Ribosome rescue allows UPR signaling from the *HAC1* 5’ exon fragment.

(A) Evaluating the role of ribosome recycling on the *ski2Δ*-dependent growth rescue of *trl1-H148Y* cells under ER stress. Serial five-fold dilutions of *trl1-H148Y* cells with the indicated deletions of *SKI2* and/or *DOM34* were spotted on YPD plates without (-Tm) or with (+Tm) 0.1 μg/ml tunicamycin and incubated at 30°C for 2 days. (B) Lysates of the indicated strains were prepared and immunoblotted for 3xHA-Hac1p and GAPDH as loading control. Full-length Hac1p is indicated by a closed arrowhead, truncated Hac1p by an open arrowhead. An even shorter minor third band is marked with an asterisk. The high molecular weight smear/banding pattern is indicated as HMW-Hac1p.

https://doi.org/10.7554/eLife.44199.012

Tables

Table 1

Data collection and refinement statistics.

https://doi.org/10.7554/eLife.44199.007

Datasets:	High-resolution (remote)	Se SAD phasing (peak)
Data collection
Beamline	APS 23ID-D	APS 23ID-D
Wavelength (Å)	1.0332	0.9793
Space group	P2₁2₁2₁	P2₁2₁2₁
Cell dimensions a, b, c (Å) α, β, γ (°)	49.73, 56.58, 173.88 90, 90, 90	49.63, 56.46, 173.87 90, 90, 90
Resolution (Å)	37.74–1.90 (1.94–1.90)	49.63–2.50 (2.60–2.50)
Total reflections	159936 (10248)	175374 (20071)
Unique reflections	39302 (2495)	17561 (1930)
Multiplicity	4.1 (4.1)	10.0 (10.4)
Completeness (%)	99.3 (99.1)	99.1 (98.1)
I/σ(I)	10.3 (1.2)	21.9 (7.3)
CC(1/2)	0.998 (0.793)	0.999 (0.982)
R_pim	0.036 (0.575)	0.024 (0.090)
Phasing
Number of Se sites		2
Figure of merit		0.64
Refinement
R_work/R_free	0.170/0.214
Number of atoms	3457
Wilson B factor (Å)	33.8
RMS deviations Bonds (Å) Angles (°)	0.007 1.386
Ramachandran plot % favored % allowed % outliers	97.91 1.83 0
PDB ID	6N67

Values in parentheses refer to the highest resolution shell.

The R_free set consists of 5% randomly chosen data excluded from refinement.

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Gene (Chaetomium thermophilum)	TRL1	Chaetomium thermophilum genome resource	Ct:CTHT_0034810
Gene (Saccharomyces cerevisiae)	TRL1	Saccharomyces Genome Database	SGD:S000003623
Gene (Saccharomyces cerevisiae)	IRE1	Saccharomyces Genome Database	SGD:S000001121
Gene (Saccharomyces cerevisiae)	HAC1	Saccharomyces Genome Database	SGD:S000001863
Antibody	anti-HA (mouse monoclonal)	Sigma	Sigma:H3663; RRID:AB_262051	(1:2000)
Antibody	anti-GAPDH (rabbit polyclonal)	abcam	abcam:ab9485; RRID:AB_307275	(1:1000)
Antibody	goat anti-mouse (goat polyclonal, HRP conjugate)	Promega	Promega:W4021; RRID:AB_430834	(1:10000)
Antibody	goat anti-rabbit (rabbit polyclonal, HRP conjugate)	Promega	Promega:W4011; RRID:AB_430833	(1:10000)
Recombinant DNA reagent	HAC1^U-508-pBSK-	PMID:9323131	lab archive:pPW0386; paper:pCF187	HAC1 in vitro transcript (508 nucleotides)
Sequence-based reagent	Cy5-5' exon HAC1 RNA oligonucleotide	IDT		5’-Cy5-CGUAAUCCAG-3’-PO4
Sequence-based reagent	Cy5-3' exon HAC1 RNA oligonucleotide	IDT		5’-PO4-AAGCGCAGUC-Cy5
Sequence-based reagent	FAM-5' exon HAC1 RNA oligonucleotide	IDT		5’-FAM-CGUAAUCCAG-3’-PO4
Sequence-based reagent	3' exon HAC1 RNA oligonucleotide	IDT		5’-PO4-AAGCGCAGUCAGGUUUGAAU-3’
Sequence-based reagent	oligo a for RNase H assay	IDT		TAATCACGGCGGACAGTA
Sequence-based reagent	oligo b for RNase H assay	IDT		TTGAAGGTACTTTAACCG
Sequence-based reagent	oligo-(dT)_12-18	Thermo Fisher Scientific	Thermo Fisher Scientific:18418012
Sequence-based reagent	random hexamers	Thermo Fisher Scientific	Thermo Fisher Scientific:N8080127
Peptide, recombinant protein	ctTrl1 [full length]	This paper		purified from E. coli BL21-RIL cells
Peptide, recombinant protein	ctTrl1-LIG	This paper		purified from E. coli BL21-RIL cells
Peptide, recombinant protein	scTrl1-LIG-WT	This paper		purified from E. coli BL21-RIL cells
Peptide, recombinant protein	scTrl1-LIG-H148Y	This paper		purified from E. coli BL21-RIL cells
Peptide, recombinant protein	scTrl1-CPD	This paper		purified from E. coli BL21-RIL cells
Peptide, recombinant protein	scIre1-KR32	This paper		purified from E. coli BL21-RIL cells
Peptide, recombinant protein	RtcA	This paper		purified from E. coli BL21-RIL cells
Peptide, recombinant protein	His₁₀-HRV-3C protease	This paper		purified from E. coli BL21-RIL cells
Peptide, recombinant protein	T4 polynucleotide kinase (3’ phosphatase minus)	New England BioLabs	New England BioLabs:M0236S
Peptide, recombinant protein	Proteinase K	Thermo Fisher Scientific	Thermo Fisher Scientific:25530049
Peptide, recombinant protein	RNAse H	New England BioLabs	New England BioLabs:M0297S
Peptide, recombinant protein	SuperScript II reverse transcriptase	Thermo Fisher Scientific	Thermo Fisher Scientific:18064014
Chemical compound, drug	AMPcPP	Sigma	Sigma:M6517
Chemical compound, drug	4μ8C	Matrix Scientific	Matrix Scientific:037985; CAS:14003-96-4
Chemical compound, drug	Tunicamycin	Sigma	Sigma:T7765
Chemical compound, drug	SYBR Gold nucleic acid stain	Thermo Fisher Scientific	Thermo Fisher Scientific:S11494