Description
Nonsense-mediated decay (NMD) is an evolutionarily conserved RNA surveillance system that degrades transcripts containing premature termination codons (Kurosaki et al., 2019).
C. elegans
SMG-1
, an ortholog of human SMG1, is a PI3K-related kinase and a key component of the NMD pathway. The role of
SMG-1
in NMD has been well characterized; it phosphorylates the
SMG-2
/UPF1 RNA helicase to promote degradation of PTC-containing transcripts (Grimson et al., 2004; Johns et al., 2007; Page et al., 1999).
SMG-1
also functions in additional pathways, including DNA double-stranded break repair (González-Huici et al., 2017; Kamp et al., 2022) and regulation of lifespan through
DAF-16
, the
C. elegans
ortholog of mammalian FOXO transcription factors (Masse et al., 2008).
The
smg-1
(
r861
)
allele was originally identified in a forward genetic screen by Hodgkin et al. (1989) as a suppressor of an
unc-54
nonsense allele.
smg-1
(
r861
)
is a strong loss-of-function allele that is defective in NMD, allowing transcripts containing PTCs to escape degradation. Despite its frequent use in studies of RNA surveillance and transgene expression, to our knowledge, the precise mutation in
smg-1
(
r861
)
has not previously been reported.
To identify the underlying mutation, we analyzed a publicly available RNA sequencing dataset containing both wild-type (Bristol
N2
) and
smg-1
(
r861
)
strains (Rubio-Peña et al., 2015). Alignment of the
smg-1
transcripts revealed a two-base pair insertion in exon 33 of the gene in
smg-1
(
r861
)
animals that was absent in the wild-type (
Figure 1A,
B). This insertion lies within the conserved PI3K/PI4K catalytic domain at residue P1846 and introduces a frameshift that results in a premature termination codon (PTC) shortly downstream of the insertion.
Interestingly, the
smg-1
transcripts remain detectable in
smg-1
(
r861
)
animals, consistent with a failure to trigger NMD. It is therefore likely that
SMG-1
kinase function is required for degradation of its own PTC-containing transcript in
smg-1
(
r861
)
mutants. However, a previous study found that
SMG-1
protein is undetectable by Western blot in
smg-1
(
r861
)
animals using an antibody directed against an epitope upstream of the PTC (Grimson et al., 2004), suggesting that the truncated transcript is either not efficiently translated or the resulting protein is rapidly degraded.
To validate the identified mutation, we amplified a 452 bp region of genomic DNA from the
smg-1
gene surrounding the predicted insertion site in wild-type and
smg-1
(
r861
)
animals. Sanger sequencing of the PCR products confirmed the presence of an AC dinucleotide insertion at Chromosome I:6,903,692 in
smg-1
(
r861
)
, which was absent in wild-type (
Figure 1C
).
Notably, this insertion creates a unique RseI restriction site that is not present in the wild-type sequence. To develop a simple genotyping assay, we digested the 452 bp PCR product with RseI. Digestion of the
smg-1
(
r861
)
amplicon produced two fragments of 397 bp and 57 bp, while the wild-type amplicon remained undigested. This approach therefore provides a rapid and reliable method for identifying the
smg-1
(
r861
)
allele based on restriction fragment length polymorphism (
Figure 1D
). We anticipate that our assay will be useful for future experiments involving crossing the
r861
allele into other relevant genetic backgrounds.
Methods
RNA seq analysis
SRA files from the GEO dataset GSE72952 were aligned to the WBcel235 reference genome using STAR (Dobin et al., 2013).
Worm maintenance
C. elegans
strains were maintained under standard conditions as described by Brenner (1974).
The following strains were used: Bristol
N2
and
TR1331
smg-1
(
r861
)
I.
All strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).
Amplification, sequencing, and digestion
Genomic DNA was extracted using a protocol based on Williams et al. (1992). Briefly, a worm lysis buffer was prepared with 50 mM Tris-HCl pH 8.0, 50 mM KCl, 2.5 mM MgCl
2
, 0.45% NP-40, 0.45% Tween 20, and 68 μg/mL proteinase K. Single adult worms were added to 6 μl lysis buffer and incubated at 65 °C for 1 hr 5 min, then 95 °C for 15 min.
Amplicons were generated with Vazyme Rapid Taq (Cat. no. P222) using 1 μl of lysed worm solution with the forward primer 5′-GTTATTCCACTTGGACCACG-3′ and reverse primer 5′-CATCCAAAGCTCACGACTG-3′. PCR was performed with an annealing temperature of 55.7 °C, 10 seconds extension, and 35 cycles. Amplicons were cleaned with Zymo Research DNA Clean & Concentrator (Cat. no. D4014).
Sanger sequencing of cleaned amplicons was performed by The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Canada.
Amplicons were digested using ThermoFisher RseI (Cat. no. ER2001) by incubating for 3 hrs at 37 °C. A 2% agarose gel in 1x TAE was run at 120 V for 60 min with the digested DNA. The gel was imaged after 500 ms UV light exposure using a Bio-Rad Gel Doc XR+ imaging system (Universal Hood II) and Image Lab software (version 6.0.1).