The survival motor neuron gene smn-1 interacts with the U2AF large subunit gene uaf-1 to regulate Caenorhabditis elegans lifespan and motor functions

Xiaoyang Gao; Yanling Teng; Jintao Luo; Liange Huang; Min Li; Zhuohua Zhang; Yong-Chao Ma; Long Ma

doi:10.4161/rna.36100

. 2014 Oct 31;11(9):1148–1160. doi: 10.4161/rna.36100

The survival motor neuron gene smn-1 interacts with the U2AF large subunit gene uaf-1 to regulate Caenorhabditis elegans lifespan and motor functions

Xiaoyang Gao ¹, Yanling Teng ¹, Jintao Luo ¹, Liange Huang ¹, Min Li ², Zhuohua Zhang ¹, Yong-Chao Ma ³, Long Ma ^1,^*

PMCID: PMC4615316 PMID: 25483032

Abstract

Spinal muscular atrophy (SMA), the most frequent human congenital motor neuron degenerative disease, is caused by loss-of-function mutations in the highly conserved survival motor neuron gene SMN1. Mutations in SMN could affect several molecular processes, among which aberrant pre-mRNA splicing caused by defective snRNP biogenesis is hypothesized as a major cause of SMA. To date little is known about the interactions of SMN with other splicing factor genes and how SMN affects splicing in vivo. The nematode Caenorhabditis elegans carries a single ortholog of SMN, smn-1, and has been used as a model for studying the molecular functions of SMN. We analyzed RNA splicing of reporter genes in an smn-1 deletion mutant and found that smn-1 is required for efficient splicing at weak 3′ splice sites. Genetic studies indicate that the defective lifespan and motor functions of the smn-1 deletion mutants could be significantly improved by mutations of the splicing factor U2AF large subunit gene uaf-1. In smn-1 mutants we detected a reduced expression of U1 and U5 snRNAs and an increased expression of U2, U4 and U6 snRNAs. Our study verifies an essential role of smn-1 for RNA splicing in vivo, identifies the uaf-1 gene as a potential genetic modifier of smn-1 mutants, and suggests that SMN-1 has multifaceted effects on the expression of spliceosomal snRNAs.

Keywords: SMA, SMN, smn-1, U2AF large subunit, uaf-1

Introduction

RNA splicing is a fundamental regulatory mechanism for eukaryotic gene expression.¹ Hundreds of proteins are found to be associated with RNA splicing.² However the in vivo functions and interactions of these proteins remain to be understood. Mutations in numerous splicing factors could lead to severe diseases.³

Spinal muscular atrophy (SMA) is caused by loss-of-function mutations in the highly conserved and ubiquitously expressed survival motor neuron (SMN) protein primarily encoded by the SMN1 gene.⁴ Humans also contain SMN2, a paralogous gene of SMN1 that is likely generated by intrachromosomal duplication.^4,5 Because of an alternative splicing event, SMN2 produces a significantly reduced level of functional SMN protein,^6-8 which might be sufficient for the survival of most cell types except that of spinal motor neurons.

The SMN protein affects proper assembly of the spliceosomal snRNPs,^9,10 the core components of the RNA splicing machinery.¹¹ In a zebrafish SMA model, the injection of purified U snRNPs into the animals could rescue motor neuron degeneration caused by induced silencing of zebrafish smn expression.¹² Defective pre-mRNA splicing was also observed in SMN-deficient S. pombe,¹³ Drosophila,^14,15 zebrafish,¹⁶ mice^16-20 and human cells.²¹ Based on these findings it was hypothesized that altered splicing of genes in SMN-deficient animals underlies the defects of these animals, including SMA.²² However it remains unknown whether restoring the splicing of any genes could substantially rescue the defects caused by SMN deficiency. In addition, it is not fully understood what features of a gene determine whether the splicing of this gene requires SMN.

Besides affecting snRNP biogenesis and RNA splicing, SMN is also believed to be required for mRNA transport in neurons,²³ axonal guidance²⁴ and muscle functions.²⁵ Therefore an alternative hypothesis proposes that disrupted mRNA transport in neurons contributes to the defects of SMN-deficient animals.²² Consistent with this hypothesis, overexpression of the protective modifier gene plastin 3 could partially rescue axonal length and outgrowth defects in SMN-deficient zebrafish and mouse motor neurons,²⁶ and could alleviate the defects of an SMA mouse model.²⁷ Recently numerous interactors of SMN have been identified in Drosophila and C. elegans.^28-30 It remains unclear how these genes interact with Drosophila and C. elegans orthologs of SMN and whether these genes contribute to the defects of SMN-deficient mutants.

We previously isolated mutations affecting the splicing factors U2AF large subunit (UAF-1) and SF1/BBP (SFA-1).³¹ We also identified a sensitive endogenous splicing reporter gene tos-1³² that can be used for analyzing in vivo regulation of alternative splicing.^32,33 The C. elegans gene smn-1 encodes an ortholog of the SMN protein and is essential for animal survival.³⁴ A deletion mutation of smn-1, ok355Δ, causes developmental arrest, a reduced lifespan and a progressive loss of motor functions,^29,35 indicating that C. elegans could serve as a valuable model for understanding the in vivo functions of SMN. It is noteworthy that reducing smn-1 expression by RNAi could lead to embryonic lethality³⁴ (X. Gao and L. Ma, unpublished observations), a defect more severe than the larval arrest of smn-1(ok355Δ) mutants.³⁵ This difference is likely caused by maternally-inherited smn-1 transcript or SMN-1 protein in smn-1(ok355Δ) mutants from smn-1(ok355Δ)/+ parental animals, which might be sufficient to support the mutants to survive into larval stages. In this study we examined how smn-1 affects tos-1 splicing in vivo, interacts with uaf-1 and affects the expression of spliceosomal snRNAs in C. elegans.

Results

A deletion mutation of smn-1 causes altered splicing of tos-1

SMN has been shown to affect RNA splicing in different species.^13-21 However it remains unknown whether SMN-1 has a similar function in C. elegans. To test this, we examined the splicing of the endogenous alternative splicing reporter gene, tos-1,³² in smn-1(ok355Δ) mutants.³⁵

Alternative splicing generally involves 4 basic modes of actions: intron retention, exon skipping (cassette exon), alternative 5′ splice sites and alternative 3′ splice sites.³⁶ The splicing of tos-1 involves intron 1 retention, exon 3 skipping and in the presence of mutations of the U2AF large subunit,³¹ the recognition of an alternative cryptic 3′ splice site in tos-1 intron 1.³² tos-1 has been used as a sensitive reporter gene for analyzing the functions of splicing factors in C. elegans.^32,33

We found that smn-1(ok355Δ) increased tos-1 intron 1 retention from 12% to about 30% (Fig. 1A and D) without an obvious effect on exon 3 skipping (Fig. 1E). We previously found that mutations in the U2AF large subunit gene uaf-1 cause recognition of a cryptic 3′ splice site in tos-1 intron 1 (Fig. 1B, dotted arrow).³² In smn-1(ok355Δ) mutants this cryptic site was not recognized (Fig. 1B and F).

Figure 1. — ***smn-1(ok355*Δ) alters the splicing of *tos-1.*** (A) RT-PCR experiments examining *tos-1* splice isoforms in different genetic backgrounds. Genotypes are labeled on top. Splice isoforms of *tos-1* are illustrated on the right. Arrows: PCR primers. (B) RT-PCR experiments examining the splicing at the cryptic 3′ splice site of *tos-1* intron 1. Genotypes are labeled on top. Splice isoforms of *tos-1* are illustrated on the right. Arrow: intron 1 endogenous 3′ splice site. Dotted arrow: intron 1 cryptic 3′ splice site. (C) RT-PCR experiments examining the splicing of *tos-1* in animals fed dsRNA targeting different splicing factor genes. Splice isoforms are illustrated on the right. (D) The molar ratios of all *tos-1* splice isoforms with intron 1 retention (isoforms 1 and 2), presented as a percentage of all isoforms combined (isoforms 1, 2, 3, 4 and 5). (E) The molar ratios of all *tos-1* splice isoforms with exon 3 skipping (isoforms 2 and 5), presented as a percentage of all isoforms combined (isoforms 1, 2, 3, 4 and 5). (F) Percentages of *tos-1* isoforms spliced at the intron 1 cryptic 3′ splice site compared to all isoforms spliced at either the endogenous 3′ splice site or the cryptic 3′ splice site. Isoform intensities were calculated by analyzing biological duplicates or triplicates using the NIH ImageJ software. Similar results for *uaf-1(n4588)* and *sfa-1(n4562)* were reported previously.³² Statistics: different from wild type (*) or control RNAi (#) or difference between the 2 compared data sets. * or #: P < 0.05 (Student's TTEST). N.S.: no significant difference. Bars: standard deviations.

We also examined tos-1 splicing in animals fed dsRNAs targeting smn-1. Using transgenic animals expressing an SMN-1(full-length)::GFP fusion protein under control of a myo-3 promoter,³⁷ we verified that 2 independent smn-1 RNAi clones obtained from 2 different C. elegans RNAi libraries,^38,39 respectively, could effectively reduce the expression of the SMN-1::GFP fusion protein as shown by GFP fluorescence (Fig. S1A) and western blot (Fig. S1B). The SMN-1::GFP fusion protein is localized in the body-wall muscle nuclei (Fig. S1A), consistent with the previous finding that the endogenous SMN-1 is expressed in the nuclei of all embryonic and young larval cells and an SMN-1(full-length)::GFP fusion protein is expressed in the nuclei of various cell types in adults.³⁴ Both smn-1 RNAi clones increased tos-1 splice isoform 2 (Fig. 1C and S1C), a product caused by both intron 1 retention and exon 3 skipping.³² Quantification of tos-1 splice isoforms verified that smn-1(RNAi) caused increased intron 1 retention and exon 3 skipping (Fig. 1D and E), an effect similar to that caused by mutations or reduced expression in uaf-1 or the splicing factor gene sfa-1.³¹ This result suggests that smn-1 is required for efficient splicing at the 3′ splice sites of both introns 1 and 2 of tos-1.

Compared to smn-1(RNAi), smn-1(ok355Δ) did not cause an obvious change of tos-1 exon 3 skipping (Fig. 1E). This is probably caused by the presence of maternally-inherited smn-1 transcript or SMN-1 protein in smn-1(ok355Δ) mutants (derived from smn-1(ok355Δ)/+ heterozygotes), which might be sufficient to support efficient splicing at the 3′ splice site of intron 2 (no exon 3 skipping) but not that of intron 1 (intron 1 retention). Such a splicing event would lead to a more abundant expression of tos-1 isoform 1 (Fig. 1A). In smn-1(RNAi) animals, smn-1 might have been depleted to a level that is not sufficient for proper splicing at either 3′ splice site of introns 1 and 2, which leads to a more abundant expression of tos-1 isoform 2 (Fig. 1C and S1C).

uaf-1(n4588) and smn-1(ok355Δ) have an additive effect on tos-1 intron 1 retention

The C. elegans gene uaf-1 encodes an ortholog of the splicing factor U2AF large subunit,⁴⁰ an essential splicing factor for the recognition of 3′ splice sites.¹¹ To understand how smn-1 and uaf-1 interact to affect splicing, we utilized a previously identified uaf-1(n4588) allele that changes a highly conserved Thr180 to Ile in the encoded UAF-1 protein.³¹ uaf-1(n4588) affects tos-1 splicing by increasing intron 1 retention and exon 3 skipping and causing recognition of a cryptic 3′ splice site in tos-1 intron 1 (Fig. 1).³² We generated smn-1(ok355Δ); uaf-1(n4588) double mutants and examined the splicing of tos-1 in these animals. The double mutants had an increased intron 1 retention compared to either smn-1(ok355Δ) or uaf-1(n4588) single mutants (Fig. 1D and S2A), suggesting that smn-1(ok355Δ) and uaf-1(n4588) have an additive effect on tos-1 intron 1 retention. The double mutants and uaf-1(n4588) single mutants had similar tos-1 exon 3 skipping (Fig. 1E and S2A), suggesting that smn-1(ok355Δ) does not enhance the increased exon 3 skipping caused by uaf-1(n4588) and consistent with the finding that smn-1(ok355Δ) alone does not affect tos-1 exon 3 skipping (Fig. 1E). Similarly, smn-1(ok355Δ) did not affect the recognition of the intron 1 cryptic 3′ splice site in the double mutants compared to uaf-1(n4588) single mutants (Fig. 1F and S2B).

smn-1 is required for efficient splicing at weak 3′ splice sites

Unlike intron 1, the splicing of tos-1 intron 3 was not affected in either smn-1(ok355Δ) or smn-1(RNAi) animals (Fig. 1 and S1). Since tos-1 intron 3 contains a strong consensus 3′ splice site (TTTTCAG) that is the most abundant 3′ splice site in the C. elegans genome,^41,42 we postulate that splicing of introns containing such a strong site in general will not be affected in smn-1(ok355Δ) mutants. We tested this hypothesis using a previously described splice site substitution experiment (Fig. 2A).³² Substituting the weak 3′ splice site of tos-1 intron 1 with TTTTCAG caused a strong splicing at this site and a complete lack of intron 1 retention (Fig. 2A, B and C, Tg-1). Similarly, substituting the weak 3′ splice site of intron 2 with TTTTCAG caused a strong splicing at the new site and a complete lack of exon 3 skipping (Fig. 2A, B and D, Tg-2). Substituting both 3′ splice sites of introns 1 and 2 with TTTTCAG resulted in a lack of both intron 1 retention and exon 3 skipping and generated a single splice isoform, isoform 4 (Fig. 2A-D, Tg-3). These results suggest that RNA splicing at the strong consensus site TTTTCAG might not require smn-1, regardless of the intron in which this site is localized.

Figure 2. — **Transgene experiments indicate that *smn-1* is required for splicing at the weak 3**′ **splice sites of *tos-1*.** (A) Illustration of the transgene structure and the substitutions of 3′ splice sites. Arrows: PCR primers for detecting transgene-specific transcripts. (B) RT-PCR experiments to detect the splice pattern of each indicated transgene in *smn-1(ok355*Δ) mutant background. Splice isoforms are indicated on the right. (C) Quantitative analysis of intron 1 retention of each transgene using the NIH Image J software. (D) Quantitative analysis of exon 3 skipping of each transgene using the NIH image J software. Statistics: different from wild type or difference between the 2 compared datasets (Student's TTEST). N.S.: no significant difference between the 2 data sets. Bars: standard deviations

uaf-1 mutations extend the lifespan of smn-1(ok355Δ) mutants

That both uaf-1 and smn-1 are required for proper RNA splicing suggests that these 2 genes might interact to affect C. elegans development and behaviors. To test this, we first examined the sizes and lifespans of the single and double mutants. We found that smn-1(ok355Δ); uaf-1(n4588) double mutants were slightly smaller than smn-1(ok355Δ) single mutants on day 3 and day 5 post the L1 larval stage (Fig. S3), suggesting that smn-1(ok355Δ) and uaf-1(n4588) have an additive effect on reducing the animal sizes. Interestingly we found that the double mutants had an apparently longer lifespan than smn-1(ok355Δ) single mutants (Fig. 3A, individual trials are shown in Fig. S4A, B and C). A higher-resolution (every 12 hrs) examination of the lifespans of these mutants indicates that uaf-1(n4588) increased the median life span of smn-1(ok355Δ) mutants from ∼7.0 days to 8.5 days post L1 (Fig. 3B, individual trials are shown in Fig. S4D and E).

Figure 3. — *uaf-1* mutations extend the lifespan of *smn-1(ok355*Δ) mutants. (A) Complete survival curves of animals at 20°C. Three independent groups of 40 to 60 animals were scored for each genotype. (B) High-resolution survival curves (every 12 hrs) at 20°C. Two independent groups of 40 to 60 animals were scored for each genotype. (C) Complete survival curves of animals at 25°C. Three independent groups of 40 to 60 animals were scored for each genotype. (D) High-resolution survival curves (every 12 hrs) at 25°C. Two independent groups of 40 to 60 animals were scored for each genotype. Statistics: difference between 2 lifespan curves. P < 0.001 (logrank test) between *smn-1(ok355*Δ) mutants and *smn-1(ok355*Δ*); uaf-1(mut)* double mutants for each individual survival trial under all experimental conditions (**see Fig. S4**).

To examine whether the effect on the lifespan of smn-1(ok355Δ) mutants is uaf-1 allele-specific, we tested a different uaf-1 allele, n4588 n5127.³¹ uaf-1(n4588 n5127) is an intragenic suppressor of uaf-1(n4588) isolated in a suppressor screen for survival of uaf-1(n4588) mutants at 25°C.³¹ uaf-1(n4588 n5127) carries a second missense mutation (M157I) in UAF-1 in addition to the original n4588 (T180I) mutation and has a similar effect on tos-1 splicing.^31,32 We found that uaf-1(n4588 n5127) similarly extended the lifespan of smn-1(ok355Δ) mutants (Fig. 3A, B).

We next tested these animals at 25°C and found that uaf-1(n4588 n5127) also increased the lifespan of smn-1(ok355Δ) mutants (Fig. 3C, individual trials are shown in Fig. S4F, G and H). A higher-resolution examination suggests a median lifespan increase from 4.25 days for smn-1(ok355Δ) single mutants to 6.75 days for smn-1(ok355Δ); uaf-1(n4588 n5127) double mutants at this temperature (Fig. 3D, individual trials are shown in Fig. S4I and S4J). [smn-1(ok355Δ); uaf-1(n4588) double mutants could not be examined at this temperature because uaf-1(n4588) causes embryonic lethality at 25°C.³¹]

uaf-1 mutations could improve the motor functions of smn-1(ok355Δ) mutants

Besides a shortened lifespan, smn-1(ok355Δ) mutants also exhibit a gradual declination of body movements and pharyngeal pumping during postembryonic development.³⁵ We therefore examined whether uaf-1 mutations could improve the motor functions of smn-1(ok355Δ) mutants.

We measured the bodybends made by the mutants in 30 sec (Fig. 4A), which was previously used as a readout for the locomotion of animals.^31,33,43 On day 3 post L1, there was no significant difference in bodybends between smn-1(ok355Δ) single mutants and smn-1(ok355Δ); uaf-1(mut) double mutants (Fig. 4A). On day 5, smn-1(ok355Δ); uaf-1(n4588) and smn-1(ok355Δ); uaf-1(n4588 n5127) double mutants had significantly improved bodybends (7.5 and 12.0, respectively) compared to smn-1(ok355Δ) single mutants (5.0 bodybends) (Fig. 4A), suggesting that uaf-1(mut) could suppress the locomotion defect of smn-1(ok355Δ) mutants.

Figure 4. — *uaf-1* mutations could improve the motor functions of *smn-1(ok355*Δ) mutants at 20°C. (A) Bodybends, (B) thrashes (C) pharyngeal pumps of animals with the indicated genotypes on day 3 and day 5 post the L1 larval stage. At least 20 animals were scored for each dataset. Statistics: difference between the 2 compared data sets. *: P < 0.01 (one-way ANOVA with Bonferroni test). Bars: standard errors.

Both smn-1(ok355Δ) single mutants and smn-1(ok355Δ); uaf-1(mut) double mutants failed to generate any bodybends on day 7. However the single mutants were mostly paralyzed along the whole body, while the double mutants were still capable of moving their heads (X. Gao and L. Ma, unpublished observations). Only on day 9 did the double mutants become completely paralyzed (X. Gao and L. Ma, unpublished observations).

To examine whether the protective effect of uaf-1(mut) on the motor functions of smn-1(ok355Δ) mutant is limited to bodybends, we scored the thrashes made by the animals in M9 as described by Briese et al.³⁵ On day 5 post L1 the thrashing rates of both smn-1(ok355Δ); uaf-1(mut) double mutants were significantly higher than that of smn-1(ok355Δ) single mutants (Fig. 4B), suggesting that uaf-1(mut) could suppress the thrashing defect of smn-1(ok355Δ) mutants.

We found that on day 3 smn-1(ok355Δ); uaf-1(4588) double mutants had a reduced thrashing rate compared to smn-1(ok355Δ) single mutants (Fig. 4B), suggesting that uaf-1 is required for this motor function of smn-1(ok355Δ) mutants at early developmental stage.

Consistent with previous findings,^29,35 we found that the pharyngeal pumping rate of smn-1(ok355Δ) mutants is about a quarter of that of the wild-type animals on day 3 post L1 (Fig. 4C). At this age, uaf-1(mut) did not affect the pumping rate of smn-1(ok355Δ) mutants (Fig. 4C). On day 5, uaf-1(n4588 n5127) significantly improved the pumping rate of smn-1(ok355Δ) mutants, while uaf-1(n4588) had no obvious effect, suggesting an allele-specific effect of uaf-1 mutations on the pharyngeal pumping of smn-1(ok355Δ) mutants.

We also measured the bodybends, thrashes and pharyngeal pumps of smn-1(ok355Δ); uaf-1(n4588 n5127) double mutants at 25°C (Fig. 5). On day 4 post L1, uaf-1(n4588 n5127) consistently improved these motor functions of smn-1(ok355Δ) mutants (Fig. 5A, B, C). In addition, smn-1(ok355Δ); uaf-1(n4588 n5127) double mutants had a better locomotion and a higher thrashing rate than uaf-1(n4588 n5127) single mutants, suggesting that smn-1(ok355Δ) could suppress the motor defects of uaf-1(n4588 n5127) mutants. On day 2 at 25°C, however, smn-1(ok355Δ); uaf-1(n4588 n5127) double mutants had a more defective thrashing rate compared to smn-1(ok355Δ) single mutants (Fig. 5B), which is similar to the thrashing result for smn-1(ok355Δ); uaf-1(n4588) double mutants on day 3 at 20°C (Fig. 4B).

Figure 5. — *uaf-1(n4588 n5127)* could improve the motor functions of *smn-1(ok355*Δ) mutants at 25°C. (A) Bodybends, (B) thrashes (C) pharyngeal pumps of animals with the indicated genotypes on day 2 and day 4 post the L1 larval stage at 25°C. At least 20 animals were scored for each dataset. Statistics: difference between the 2 compared data sets. *: P < 0.01 (one-way ANOVA with Bonferroni test). Bars: standard errors.

uaf-1(n4588) causes in UAF-1 both a loss of function that reduces the recognition of several weak 3′ splice sites and an altered function that leads to enhanced recognition of certain weak 3′ splice sites.^31,32 We found that reducing uaf-1 expression³¹ by RNAi feeding did not obviously improve the locomotion (Fig. S5A) or extend the lifespan (Fig. S5B) of smn-1(ok355Δ) mutants, suggesting that probably the altered UAF-1 function, but not the loss of UAF-1 function, reversed smn-1(ok355Δ) defects. Since uaf-1(n4588 n5127) is a weaker allele of uaf-1(n4588) and exhibits a similar effect on alternative splicing,^31,32 we suggest that uaf-1(n4588 n5127) might reverse smn-1(ok355Δ) mutant defects by a similar mechanism.

smn-1 is differentially required for the expression of spliceosomal snRNAs

Previous studies suggest that SMN affects the biogenesis of spliceosomal snRNPs in human,²¹ mouse^19,44 and yeast.¹³ The expression levels of spliceosomal U snRNAs are generally reduced in SMN mutants of these species. We therefore examined whether C. elegans smn-1 has a similar effect on the expression of U snRNAs.

Using qRT-PCR, we quantified the expression levels of U1, U2, U4, U5 and U6 snRNAs (Fig. 6) and found that smn-1(ok355Δ) differentially affected the expression of these snRNAs. For example, the expression levels of U1 and U5 were both reduced on days 3 and 5 post L1 in smn-1(ok355Δ) mutants (Fig. 6A and B). However the expression level of U4 was not significantly different from that of wild-type animals on day 3 (Fig. 6A) and was apparently increased on day 5 (Fig. 6B). Different from that of U1, U4 and U5, the level of U2 in smn-1(ok355Δ) mutants was apparently increased compared to that of wild-type animals on both days 3 and 5 (Fig. 6A and B). The most striking difference was observed for U6, the level of which was over 24 folds of that of wild-type animals on day 3 (Fig. 6A) and was increased to over 200 folds on day 5 (Fig. 6B). We verified the dramatic change of U6 expression by performing qualitative RT-PCRs and examining the amplified U6 cDNA fragments on agarose gels (Fig. S6).

Figure 6. — **qRT-PCR quantification of U snRNA expression.** Normalized fold expression of U snRNAs on (A) day 3 and (B) day 5 post the L1 larval stage using 18S rRNA as a loading control. Values are averages of biological duplicates. Statistics: different from wild type or difference between the 2 compared datasets. **: P < 0.01, *: P < 0.05 (Student's TTEST). Bars: standard errors.

Under all circumstances, smn-1(ok355Δ); uaf-1(n4588) double mutants had a similar or reduced expression of each of the 5 U snRNAs compared to smn-1(ok355Δ) single mutants (Fig. 6). For example, the expression levels of U1 and U2 on both days 3 and 5, of U4 on day 3, and of U5 on day 5 were indistinguishable between smn-1(ok355Δ) single mutants and smn-1(ok355Δ); uaf-1(n4588) double mutants (Fig. 6). Compared to smn-1(ok355Δ) single mutants, smn-1(ok355Δ); uaf-1(n4588) double mutants had a slightly reduced expression of U4 on day 5 and of U5 on day 3, and an apparently reduced expression of U6 on both days 3 and 5 (Fig. 6).

It is interesting to note that uaf-1(n4588) itself had no apparent effect on the expression of most U snRNAs, except that it reduced the expression of U1 on day 3 (Fig. 6A and B). Therefore the suppression of smn-1(ok355Δ) mutant defects by uaf-1(n4588) is likely not caused by increased U snRNA expression.

The expression of C. elegans plastin 3 (PLS3) ortholog plst-1 is significantly reduced in smn-1(ok355Δ) mutants

Plastin 3 (PLS3) was previously identified as a protective modifier of human SMA patients.²⁶ Overexpression of PLS3 could alleviate the defects of a mouse model of SMA.²⁷ The C. elegans gene plst-1 is an ortholog of PLS3 and could interact with smn-1 to affect the body length and pharyngeal pumping rate of an animal.²⁹ We found that smn-1(ok355Δ) reduced plst-1 expression to less than 20% of the wild-type level on days 3 and 5 post L1 (Fig. 7A). uaf-1(n4588) also apparently reduced plst-1 expression (Fig. 7A). However smn-1(ok355Δ); uaf-1(n4588) double mutants had a similar plst-1 expression level compared to smn-1(ok355Δ) single mutants (Fig. 7A), suggesting that smn-1(ok355Δ) and uaf-1(n4588) do not have an additive effect on reducing plst-1 expression.

Figure 7. — **Expression analyses of *C. elegans plst-1* gene and *stasimon* homologs.** (A) qRT-PCR analysis of *plst-1* expression in different mutants. Values are averages of biological replicates. Statistics: different from wild type and difference between the 2 compared data sets. *: P < 0.05, **: P < 0.01 (Student's TTEST). Bars: standard errors. (B) The *C. elegans* genome contains 3 genes encoding proteins homologous to Stasimon. The sequence identities between the *C. elegans* homologs and the human Stasimon are indicated. (C) Qualitative RT-PCR analyses of the full-length transcripts of the *C. elegans stasimon* homologs. Two biological replicates of the indicated genotypes were analyzed for each gene.

Recently the splicing of the minor intron-containing gene stasimon is found to be altered in SMN-deficient Drosophila and NIH 3T3 cells¹⁴ and stasimon overexpression could partially rescue neuronal defects in SMN-deficient Drosophila and zebrafish.¹⁴ The C. elegans genome has 3 stasimon homologs (Fig. 7B, BLAST search) that do not contain minor introns (www.wormbase.org). We examined the full-length transcripts of these genes by qualitative RT-PCR and failed to identify obvious differences among wild-type animals, smn-1(ok355Δ); uaf-1(n4588) double mutants and either of the single mutants (Fig. 7C). Therefore C. elegans stasimon homologs might not require SMN-1 for splicing.

Discussion

In this study, we provide in vivo evidence that C. elegans smn-1 is required for proper RNA splicing and the expression of spliceosomal snRNAs. We also suggest that mutations of the U2AF large subunit gene uaf-1 might be protective modifiers of smn-1 loss-of-function mutants. Together with previous studies of other SMN modifiers,^18,26-30,45 our findings should facilitate the understanding of SMN functions.

Many genes with altered splicing have been identified in SMN-deficient mutants.^13-21 However it remains unclear what cis features determine the involvement of SMN in the splicing of these transcripts. Campion et al¹³ found that increasing the length of intronic polypyrimidine tracts could improve the splicing efficiency of target introns in SMN-deficient S. pombe. We found that in C. elegans the splicing of 2 weak 3′ splice sites is dependent on smn-1 while that of a strong consensus site is not as sensitive. Therefore our results suggest a conserved function of SMN for efficient splicing of weak introns, which are characterized by the presence of short intronic polypyrimidine tracts in S. pombe⁴⁶ or weak 3′ splice sites in C. elegans.⁴¹

Of the numerous genes exhibiting altered expression and/or splicing in SMN-deficient mutants, little is known about the contributions of these genes to the mutant defects. Our findings could facilitate the characterization of orthologs of such genes. For example, compared to smn-1(ok355Δ) mutants, defective splicing that is restored in smn-1(ok355Δ); uaf-1(mut) double mutants might contribute to the defects. We found that the splicing of tos-1 is altered by smn-1(ok355Δ) (Fig. 1). However, tos-1 splicing is altered more in smn-1(ok355Δ); uaf-1(n4588) double mutants than it is in smn-1(ok355Δ) single mutants Fig. S2), suggesting that altered tos-1 splicing likely does not contribute to the smn-1(ok355Δ) mutant defects and is not a reliable indicator of the lifespans and motor functions of these animals. We postulate that expression changes of unknown genes might serve as better indicators for the lifespans and motor functions of these animals.

In smn-1(ok355Δ) mutants we detected a significantly reduced expression of plst-1, the C. elegans ortholog²⁹ of the mammalian SMA protective modifier plastin 3 (PLS3).^26,27 (Fig. 7A) Compared to smn-1(ok355Δ) single mutants, smn-1(ok355Δ); uaf-1(n4588) double mutants did not have an obviously increased expression of plst-1, suggesting that compensating plst-1 expression might not underlie the improved lifespan and motor functions of the double mutants.

In smn-1(ok355Δ) mutants we also failed to identify obviously defective splicing of the 3 C. elegans homologs of stasimon (Fig. 7B), a minor intron-containing gene identified in Drosophila and human, which by overexpression can partially rescue the mutant phenotypes of SMN-deficient Drosophila and zebrafish.¹⁴ As a genome-wide survey suggests that minor introns do not exist in C. elegans,⁴⁷ it is likely that defective minor intron splicing in general does not contribute to C. elegans smn-1(ok355Δ) mutant defects. Our results are consistent with other recent findings suggesting that altered minor intron splicing might not contribute to the developmental defects of SMN-deficient Drosophila and mouse.^15,20,48

The Drosophila Smn mutants exhibit an arrested development,^48,49 raising the question whether a more severely arrested development accounts for the extended lifespan and improved motor functions of smn-1(ok355Δ); uaf-1(mut) double mutants. However 2 pieces of evidence suggest otherwise. First, the C. elegans clk-1 (clock gene) mutants have a delayed development that results in an extended lifespan, a reduced pharyngeal pumping and a slower locomotion,⁵⁰ which are different phenotypes compared to that of smn-1(ok355Δ); uaf-1(mut) double mutants. Second, we found that smn-1(ok355Δ) single mutants had a similar gonad development as smn-1(ok355Δ); uaf-1(n4588 n5127) double mutants did (Fig. S7), suggesting that these mutants have a similar postembryonic development.

The effect of SMN on the expression levels of major spliceosomal snRNAs has been variable, exhibiting both tissue-specific^19,21,44 and species-specific effects.^{13,15,19,25,44,51} We identified an effect of smn-1 on the expression of spliceosomal snRNAs that appears to be partially consistent with previous findings. For example we found that smn-1(ok355Δ) caused reduced expression of the U1 and U5 snRNAs, consistent with observations made in mice,^19,44 Drosophila¹⁴ and S. pombe.¹³ However the expression of U4 snRNA in smn-1(ok355Δ) mutants was not affected on day 3 and was apparently increased on day 5 post L1, which is different from previous studies. Furthermore the expression levels of U2 and U6 snRNAs were apparently increased in smn-1(ok355Δ) mutants. Specifically the increase of U6 snRNA expression is striking, which was over tens (on day 3 post L1) or hundreds (on day 5 post L1) of folds of that of wild-type animals. Consistent with our findings, it has been shown that the expression levels of U2 and U6 snRNAs are also up-regulated in the kidney and skeletal muscle of an SMN mutant mouse model.¹⁹ We propose that SMN could have a conserved function in repressing the expression of U2 and U6 snRNAs in a tissue- and species-specific manner.

It remains unsolved whether the expression levels of spliceosomal snRNAs reflect the biogenesis of snRNPs in smn-1(ok355Δ)-containing mutants. We have not been able to examine the levels of snRNPs in these mutants as the animals arrest in larval stages and have to be picked individually, which significantly limited the amount of animals we could collect for reliable protein expression analysis.

We found that uaf-1(mut) caused behavioral defects to smn-1(ok355Δ) mutants at early developmental stage (when maternally inherited smn-1 level is likely high) while suppressed these defects in smn-1(ok355Δ) mutants at late stage (when maternally inherited smn-1 level is likely low), suggesting that uaf-1(mut) might be detrimental when smn-1 expression is abundant but becomes beneficial when smn-1 expression is diminished. Our findings bear similarity to previous analyses of other genetic modifiers of smn-1, in which these modifiers affect the phenotypes of control and smn-1 mutants differently.²⁹ For example, reducing the expression of the kcnl-2 gene (encoding an SK channel subunit) causes defective growth in control animals (detrimental) but suppresses the pharyngeal pumping defects of smn-1(ok355Δ) mutants (beneficial),²⁹ while a plst-1 deletion mutation promotes the growth of control animals (beneficial) but enhances the pharyngeal pumping defects of smn-1(ok355Δ) mutants (detrimental).²⁹ Our result provides a new piece of evidence that altered RNA splicing underlies SMN mutant defects, consistent with the notion that RNA processing/translational control and endocytosis are 2 pathways of particular importance for SMA pathology.^22,29

It is noteworthy that our study does not exclude the possibility that closely linked background mutations near the loci of smn-1 and/or uaf-1 could affect the phenotypes of the mutants. Specifically, uaf-1(n4588 n5127) is an intragenic suppressor derived from uaf-1(n4588),³¹ and could carry the same background mutations closely linked to the uaf-1(n4588) locus. In addition, the severe defects of smn-1(ok355Δ) were not fully rescued by neuronal- or muscle-specific expression of smn-1 transgenes,³⁵ leaving open the possibility that background mutations might contribute to the mutant phenotypes. Therefore further studies are warranted to corroborate our genetic findings and uncover the underlying molecular details.

In short, we propose that mutations in uaf-1 might act as protective modifiers of smn-1 mutants and suggest that SMN-1 has conserved functions in affecting RNA splicing and snRNA expression in C. elegans. Future analyses might reveal how uaf-1 and smn-1 interact to affect C. elegans development and behaviors.

Materials and Methods

Strains

C. elegans strains were grown at 20°C as described, unless otherwise indicated.⁵² N2 (Bristol) was the reference wild‑type strain. Strains used in this study include:

LM99 smn-1(ok355Δ) I/hT2[bli-4(e937) let-?(q782) qIs48](I,III),³⁵

MT16492 uaf-1(n4588) III,³¹

MT17922 uaf-1(n4588 n5127) III,³¹

MT14725 sfa-1(n4562) IV/nT1[qIs51](IV,V),³¹

MT20415 smn-1(ok355Δ) I/hT2[bli-4(e937) let-?(q782) qIs48](I,III); uaf-1(n4588) III,

CSM282 smn-1(ok355Δ) I/hT2[bli-4(e937) let-?(q782) qIs48](I,III); uaf-1(n4588 n5127) III,

CSM30, CSM70 smn-1(ok355Δ) I/hT2[bli-4(e937) let-?(q782) qIs48](I,III); macEx80, macEx85[tos-1(Tg-wt)],³²

CSM51, CSM81 smn-1(ok355Δ) I/hT2[bli-4(e937) let-?(q782) qIs48](I,III); macEx84, macEx88[tos-1(Tg-1)],³²

CSM82, CSM83 smn-1(ok355Δ) I/hT2[bli-4(e937) let-?(q782) qIs48](I,III); macEx89, macEx90[tos-1(Tg-2)],³²

CSM71, CSM80 smn-1(ok355Δ) I/hT2[bli-4(e937) let-?(q782) qIs48](I,III); macEx86, macEx87[tos-1(Tg-3)],³²

CSM398, CSM399, CSM400 macEx265, macEx266, macEx267[myo-3p::GFP],

CSM401, CSM402, CSM403 macEx268, macEx269, macEx270[myo-3p::smn-1gDNA(full-length)::GFP].

RT-PCR experiments

Total RNA was prepared using Trizol (Invitrogen), treated with RNase-Free DNase I (New England Biolabs) and incubated at 75°C for 10 min to inactivate DNase I. First‑strand cDNA was synthesized with random hexamer oligonucleotides using the Superscript III First-Strand Synthesis Kit (Invitrogen).

Since the splicing of tos-1 is not affected by developmental stages,³³ we used animals of mixed developmental stages for the RT-PCR experiments (Figs. 1 and 2 and S2). For each strain 2 to 3 biological replicates were analyzed and the proportion of each splice isoform was quantified using the NIH ImageJ software as described.³² Student's TTEST was used to compare the difference between 2 datasets.

smn-1(ok355Δ) homozygous animals arrest at late larval stages³⁵ and smn-1(ok355Δ) is maintained as a balanced allele using the hT2 chromosomal translocation (genotype: smn-1(ok355Δ)/hT2[qIs48]; +/hT2[qIs48]).³⁵ We generated tos-1 transgenic smn-1(ok355Δ)/ hT2[qIs48]; +/hT2[qIs48] heterozygous lines and picked transgenic smn-1(ok355Δ) mutants individually under a fluorescence microscope for RNA analysis. For each transgenic line, we picked 500 to 800 body-wall muscle GFP-positive (caused by the tos-1 transgene coinjection marker myo-3p::GFP (pPD95.86::GFP), which expresses GFP in the body-wall muscles under the control of a myo-3 promoter) and pharyngeal GFP-negative (smn-1(ok355Δ) homozygous) animals aged days 3 to 5 post L1. Two stable lines were established and analyzed for each tos-1 transgene. Proportions of intron 1 retention or exon 3 skipping were calculated as the combined ratios of all transcripts with intron 1 retention or exon 3 skipping, respectively, as described.³² Student's TTEST was used to compare the difference between 2 data sets.

For U snRNAs and plst-1 qRT-PCR experiments, animals were synchronized by bleaching and allowed to grow for 3 or 5 days post the L1 larval stage. For wild-type and uaf-1(n4588) animals, 2 independent samples of total RNA were prepared from sufficient numbers of animals. For smn-1(ok355Δ) and smn-1(ok355Δ); uaf-1(n4588) genotypes, 2 independent samples of ～1000 homozygous mutants were picked under a fluorescence dissecting microscope. ∼0.5 μg total RNAs from each sample were used for RT experiments as described above. qPCRs were performed in 3 technical replicates using the Maxima SYBR Green qPCR Master Mix (Thermo Scientific). The reaction mix contains 2 μl of diluted template (derived from ∼0.1 ng total RNA) and 0.47 μM of each primer in a final volume of 15 μl. Fluorescence signals were detected using Bio-Rad CFX96 real-time cycler with the following cycling conditions: 10 min at 95°C, followed by 40 2-step cycles of 15 s at 95°C, 30 s at 60°C. Melting curves were analyzed for each reaction and each curve contained a single peak. 18S rRNA is the reference gene. The expression level of each gene was quantified using Bio-Rad CFX Manager 3.0 and the ΔΔCt method⁵³. The average fold changes and standard errors were quantified from 2 biological replicates and Student's TTEST was used to compare the difference between 2 datasets. PCR primers are listed in Table S1.

cDNAs of stasimon homologs were prepared as described above from synchronized animals aged 3 days post L1. PCR experiments for stasimon homologs were performed as following: 94°C for 20 sec, annealing temperature for 20 sec, 72°C for 1 min, 40 cycles, followed by 72°C for 5 min. Annealing temperatures for bus-19, Y71A12C.2 and tag-175 are 58°C, 60°C and 56°C, respectively. PCR for act-1 cDNA (as the loading control) was performed as following: 94°C for 20 sec, 58°C for 20 sec, 72°C for 30 sec, 32 cycles, followed by 72°C for 5 min.

Plasmids

tos-1 transgene constructs were described before.³² For the myo-3p::smn-1::GFP transgene, a full-length smn-1 gDNA was amplified from wild-type total gDNAs and inserted into a BamHI/AgeI site of the pPD93.97 construct in-frame with GFP. myo-3p::smn-1::GFP drives the expression of a nuclear SMN-1::GFP fusion protein in the bodywall muscles (Fig. S1A), which can be detected using a rabbit anti-GFP antibody (Cell Signaling Technology, No. 2956) (Fig. S1B). As a loading control, C. elegans tubulin was detected using a mouse anti-α-tubulin antibody (Abcam, ab7291).

Behavioral assay

Eggs were released by bleaching and incubated in M9 overnight with shaking to generate synchronized L1 animals. On day 3 (20°C) or day 2 (25°C) post L1, animals were picked under a fluorescent dissecting microscope (Olympus SZX16) and transferred to a fresh OP50-seeded NGM plate. Bodybends of at least 20 animals were measured for 30 sec as described⁴³ and were measured again after 2 days. For animals treated with RNAi, large F₁ progeny of similar sizes were transferred to a fresh RNAi-feeding plate on day 5 post the treatment, and bodybends were measured on next day (day 6) and the day after (day 7), respectively.

For thrashing assay, animals were transferred to M9, allowed to adapt for 5 min and measured for one minute by counting 2 bends as one thrashing.

For pharyngeal pumping, terminal bulb pumps of animals were measured under a dissecting microscope for one minute.

For animals grown at 25°C, bodybends, thrashes and pharyngeal pumps were measured on days 2 and 4 post the L1 larval stage, respectively, as described above.

One-way ANOVA with Bonferroni test was used to compare the multiple data sets.

Body length measurement

Animals were washed from NGM plate with 20 mM NaN₃, allowed to settle in an eppendorf tube, and transferred to an unseeded NGM plate. Photomicrographs of the paralyzed animals were taken using an Olympus DP72 digital camera connected to an Olympus SZX16 dissecting microscope. Animal length was measured using the Olympus software DP2-BSW along the midline from the beginning of the head to the base of the tail. The average length of young adult N2 animals is assigned as 1 mm. Student's TTEST was used to compare the difference of 2 datasets.

Lifespan measurement

Synchronized animals were picked to a fresh OP50-seeded NGM plate on day 3 (20°C) or day 2 (25°C) post the L1 larval stage. The survival of the animals was determined either every 2 days (with 50 μM 5-fluorodeoxyuridine to prevent the hatching of F₁ progeny) or every 12 hours (no FUDR added) by observing the locomotion, head movement, pharyngeal pumping or slight movement in any parts of the body. Animals that did not move and failed to respond to touches were scored dead. Logrank test was used to compare the difference of lifespans of different strains.

RNA interference

Young adult animals were fed HT115 (DE3) bacteria containing plasmids expressing dsRNAs targeting uaf-1, sfa-1 or smn-1 on NGM plates with 1 mM IPTG and 0.1 mg/ml Ampicillin⁵⁴ for 5 days. F₁ progeny, which were arrested at different developmental stages, were washed from the RNAi plates, rinsed with H₂O and used for preparing total RNAs using Trizol (Invitrogen). The RNAi feeding bacterial strains for uaf-1 and sfa-1 were described before.³¹ The RNAi feeding bacterial strains for smn-1 were obtained from a whole-genome RNAi library³⁸ and an ORFeome RNAi library,³⁹ and the sequences of the plasmids were determined to confirm the presence of smn-1 coding exons.

Transgene experiments

Germline transgene experiments were performed as described.⁵⁵ Transgene mixtures generally contained 20 μg/ml 1 kb DNA plus ladder (Invitrogen), 20 μg/ml S. cerevisiae genomic DNA, 20 μg/ml myo-3p::GFP (pPD95.86::GFP) as a co-injection marker and 10 μg/ml of the transgene of interest.

Supplementary Material

Supplemental Material

krnb-11-09-977145-s001.zip^{(3.7MB, zip)}

Author Contributions

XG and LM designed the experiments. XG, YT, JL, LH and LM performed the experiments. XG and LM analyzed the data. ML, ZZ, YM and LM wrote the manuscript.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Bob Horvitz for providing reagents. We thank members of the Ma laboratory for suggestions. We thank Andy Fire for providing the pPD plasmids. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

Funding

This study was supported by a National Key Basic Research Program of China grant (2011CB510005), an NSFC grant (31371253) and a Projects of International Cooperation and Exchanges NSFC grant (81161160571) to LM.

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Supplemental Material

krnb-11-09-977145-s001.zip^{(3.7MB, zip)}

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PERMALINK

The survival motor neuron gene smn-1 interacts with the U2AF large subunit gene uaf-1 to regulate Caenorhabditis elegans lifespan and motor functions

Xiaoyang Gao

Yanling Teng

Jintao Luo

Liange Huang

Min Li

Zhuohua Zhang

Yong-Chao Ma

Long Ma

Abstract

Introduction

Results

A deletion mutation of smn-1 causes altered splicing of tos-1

Figure 1.

uaf-1(n4588) and smn-1(ok355Δ) have an additive effect on tos-1 intron 1 retention

smn-1 is required for efficient splicing at weak 3′ splice sites

Figure 2.

uaf-1 mutations extend the lifespan of smn-1(ok355Δ) mutants

Figure 3.

uaf-1 mutations could improve the motor functions of smn-1(ok355Δ) mutants

Figure 4.

Figure 5.

smn-1 is differentially required for the expression of spliceosomal snRNAs

Figure 6.

The expression of C. elegans plastin 3 (PLS3) ortholog plst-1 is significantly reduced in smn-1(ok355Δ) mutants

Figure 7.

Discussion

Materials and Methods

Strains

RT-PCR experiments

Plasmids

Behavioral assay

Body length measurement

Lifespan measurement

RNA interference

Transgene experiments

Supplementary Material

Author Contributions

Disclosure of Potential Conflicts of Interest

Acknowledgments

Funding

References

Associated Data

Supplementary Materials

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