Multipotent Genetic Suppression of Retrotransposon-Induced Mutations by Nxf1 through Fine-Tuning of Alternative Splicing

doi:10.1371/journal.pgen.1000484

Journal List > PLoS Genet > v.5(5); May 2009

PLoS Genet. 2009 May; 5(5): e1000484.

Published online 2009 May 15. doi: 10.1371/journal.pgen.1000484.

PMCID: PMC2674570

Copyright Concepcion et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Multipotent Genetic Suppression of Retrotransposon-Induced Mutations by Nxf1 through Fine-Tuning of Alternative Splicing

Dorothy Concepcion,^1,^2,³ Lisbeth Flores-García,^1,^2,³ and Bruce A. Hamilton^1,^2,³^*

¹Department of Medicine, University of California San Diego School of Medicine, La Jolla, California, United States of America

²Department of Cellular and Molecular Medicine, University of California San Diego School of Medicine, La Jolla, California, United States of America

³Rebecca and John Moores UCSD Cancer Center, University of California San Diego School of Medicine, La Jolla, California, United States of America

Wayne N. Frankel, Editor

The Jackson Laboratory, United States of America

* E-mail: bah/at/ucsd.edu

Conceived and designed the experiments: DC LFG BAH. Performed the experiments: DC LFG BAH. Analyzed the data: DC LFG BAH. Wrote the paper: DC LFG BAH.

Received March 25, 2009; Accepted April 17, 2009.

Abstract

Cellular gene expression machinery has coevolved with molecular parasites, such as viruses and transposons, which rely on host cells for their expression and reproduction. We previously reported that a wild-derived allele of mouse Nxf1 (Tap), a key component of the host mRNA nuclear export machinery, suppresses two endogenous retrovirus-induced mutations and shows suggestive evidence of positive selection. Here we show that Nxf1^CAST suppresses a specific and frequent class of intracisternal A particle (IAP)-induced mutations, including Ap3d1^mh2J, a model for Hermansky-Pudlak syndrome, and Atcay^hes, an orthologous gene model for Cayman ataxia, among others. The molecular phenotype of suppression includes ~two-fold increase in the level of correctly-spliced mRNA and a decrease in mutant-specific, alternatively-processed RNA accumulating from the inserted allele. Insertional mutations involving ETn and LINE elements are not suppressed, demonstrating a high degree of specificity to this suppression mechanism. These results implicate Nxf1 in some instances of pre-mRNA processing, demonstrate the useful range of Nxf1^CAST alleles for manipulating existing mouse models of disease, and specifically imply a low functional threshold for therapeutic benefit in Cayman ataxia.

Author Summary

Retroviruses and transposable elements are molecular parasites that integrate into the host genome and require host cell machinery for gene expression, replication and dissemination. Integrating elements can alter the expression of nearby host genes through both transcriptional and post-transcriptional mechanisms. Components of the host cell machinery that can adapt to favor genetic programs of the host cell over those of the parasite may afford one level of innate immunity. In laboratory mice, endogenous retroviruses are virus-derived mobile elements that account for many spontaneous mutations. A frequent class involves retrotransposition into introns of genes in the transcriptional sense orientation, which alters host gene pre-mRNA splicing. Here we show that for the intracisternal A particle (IAP) family of endogenous retroviruses, an allele of the canonical mRNA export factor Nxf1 found in wild Asiatic mice (Mus musculus castaneus) suppresses most insertions of this class (six of seven tested). To our knowledge, these results make Nxf1 the most broadly interacting modifier gene yet documented in this well-studied species. These results have significant implications for manipulating gene expression in mouse models of disease, the role of Nxf1 in pre-mRNA processing and in the dynamic range for therapeutic intervention in Cayman ataxia.

Introduction

Retroviruses and transposable elements both utilize host cell factors for their own expression and influence the expression of adjacent host genes through a variety of mechanisms. Components of host cell gene regulatory machinery that interact with molecular parasites may be regarded as components of innate immunity if they can discriminate between host and parasite expression [1]. The generality and exploitability of any given mechanism is an important practical question. Nuclear-cytoplasmic export of RNA is an important point of contact between molecular parasites and host genomes that may fit this criterion for several molecular parasites in mice and humans [2],[3]. We have previously reported that a wild-derived allele of Nxf1, which encodes the major mRNA nuclear export factor, can significantly suppress two mutations caused by insertions of endogenous retroviruses into introns of cellular genes by modulating their mature transcript levels ~2 fold [4]. A 16 kb transgene containing the full Nxf1 haplotype, but no other recognized gene, was able to confer the modifier phenotype. Whether this interaction could be generalized to a broader class of insertional events, and if so for what range of insertions, was limited by the relatively small number of events examined.

Nxf1 (also called Tap) was first described as a cellular factor that interacts with the Tip protein of herpesvirus saimiri [5] and subsequently shown to be an essential host factor for nuclear export of unspliced viral genomes of simple retroviruses [6]. Although recruitment of Nxf1 to cellular mRNPs may generally be mediated by protein contacts [7],[8], both Nxf1 and its yeast homolog Mex67p also bind RNA directly [9]–[11]. In mammals, known direct targets of Nxf1 include both exogenous and endogenous viral RNAs as well as host sequences [6], [12]–[14]. In addition, we previously reported that one Nxf1 haplotype shows hallmarks of recent positive selection in wild Mus musculus castaneus accessions [4], which may suggest a host-pathogen interaction mediated by Nxf1 in wild populations.

Endogenous retroviruses (ERVs) are non-infectious molecular parasites that are frequent mutagens in mice. Several families of ERV are highly polymorphic among classical inbred strains and among wild accessions [15]. In laboratory mice, ERV insertions account for 10–15% of spontaneous mutations [16],[17], depending on the strains from which estimates are drawn. The intracisternal A particle (IAP) and MusD/early transposon (ETn) families of ERV, which account for most of these, have different apparent rates of transposition in different inbred strains: IAPs appear to be particularly active in C3H strains and ETn elements in A strains [16]. Characteristics of autonomously active copies have been described [18]. Interestingly, the size distribution for newly integrated ETn elements is both broader and, on average, a lower percentage of full length than for IAP elements [19]. As both families are thought to have derived originally from infectious viruses, mechanisms that regulate ERVs or mitigate their impact on host genomes may have broader implications for both gene expression and host-parasite interactions.

To test the range of insertion events for which the modifier activity of Nxf1^CAST is effective, we examined gene expression, visible phenotypes, or both for five additional IAP, one LINE, and seven ETn insertion alleles. The host genes cover a wide range of phenotypes, expression patterns, and biochemical pathways:

The genes mutated in classical coat color mutations mahogany (Atrn) and mahoganoid (Mgrn) both mediate intercellular signaling by secreted agouti protein. Atrn encodes a transmembrane accessory receptor [20],[21], while Mgrn encodes an E3 ubiquitin-protein ligase that participates in endosomal trafficking [22]. Spontaneous alleles at either gene range in effect from modest coat color changes to spongiform neurodegeneration with associated neurological deficits [20],[21],[23],[24]. Among these alleles, Atrn^mgL and Mgrn^md are de novo IAP insertions into introns in the transcriptional sense orientation [24],[25] that decrease the steady-state level of correctly processed mRNA in mutant tissues, resulting in moderate coat color darkening, but lacking the neurodegeneration seen in stronger alleles.
Spontaneous mocha alleles of the intracellular trafficking adapter protein gene Ap3d1 [26] include a hypomorphic IAP insertion allele (mh^2J) that reduces levels of wild-type RNA and protein. In addition to coat color dilution caused by sorting defects in melanosomes, mh^2J and more severe alleles show substantial mortality, neurological and behavioral impairments [27]. Because mutations in other Ap3 complex proteins are associated with Hermansky-Pudlak syndrome, mocha mice have been used to model this disease [26],[27].
The ataxia^J mutation is an IAP insertion into an intron of the ubiquitin specific protease gene Usp14. Although the protein targets have not been systematically identified, loss of Usp14 activity results in synaptic defects that manifest behaviorally as tremor and ataxic gait in Usp14^axJ mice [28].
The classical mouse locus jittery is orthologous to the CRAL-TRIO domain gene ATCAY mutated in human Cayman ataxia [29]. Patients with this recessive disorder have a prominent but non-progressive psychomotor impairment consistent with cerebellar disease [30]. The hesitant mutation (Atcay^hes ) is an IAP insertion into the first intron, resulting in profound locomotor deficits with no obvious neuroanatomical correlates [29].
Mutations of the Mitf transcription factor gene block melanocyte development, causing white-spotting and other defects in mice and Waardenburg syndrome in humans. The mouse black-eyed white allele is a L1 LINE element inserted into an intron that disrupts splicing of one alternative 5′ exon [31]. Loss of this isoform results in recessive severe white spotting, such that the fur is most often completely white, with pigmented patches occurring in some animals. Weaker alleles of Mitf show larger and more frequent area of pigmented fur, providing a sensitive phenotypic readout for allele strength and modifier genes [32].
The MusD/ETn family are endogenous retroviruses that are more closely related to the IAP superfamily than most other currently active mouse retroelements [33],[34]. BALB/cJ and A/J strains carry several recent MusD/ETn family insertions that are mutagenic with respect to host genes [19]. In particular, Zhx2 is a transcriptional repressor required to down-regulate expression of Afp fetal globin RNA. Loss of Zhx2 expression in BALB/cJ (but not other BALB/c lines) due to an ETn-II insertion results in persistent Afp expression into adulthood [35],[36]. Insertion of an ETn in the gene encodng dysferlin, Dysf, in A/J mice results in loss of expression and creates an orthologous gene model for human limb-girdle muscular dystrophy 2B [37].

Here we show that Nxf1^CAST suppresses six of six IAP insertions of the IΔ1 class [38], the most frequent class of new insertions, but does not suppress a full-length IAP, a L1-LINE, nor any of six ETn insertion mutations. We quantify RNA and protein levels to show a consistent ~2-fold increase in normal gene expression from the mutant allele in each case of suppression. Concomitant decrease in the expression of mutant-specific RNAs implicates Nxf1 in pre-mRNA processing in addition to its known role in mRNA export. For disease models and other mouse mutations induced by IAP-IΔ1 retrotransposition, Nxf1^CAST provides a genetic rheostat for gene activity in situ.

Results

Nxf1^CAST Suppresses RNA Expression Phenotypes of Mgrn^md, but Not Atrn^mgL

To test whether Nxf1^CAST can suppress the RNA processing defects in Atrn^mgL and Mgrn^md, we examined whole brain RNA of progeny from genetic crosses to Nxf1^CAST, comparing homozygous mutant littermates that differ in Nxf1 genotype. Because each of these crosses also segregated other loci contributing to coat color, we did not assess pigmentation phenotypes for these two mutants.

For Atrn (Figure 1

), abnormally processed message from mg^L alleles are detected on Northern blots by probes containing exons 5′ to the insertion site, but not by the 3′ untranslated region ([25] and Figure 1A, B

). Because the large but low-abundance normally spliced message was difficult to quantify reliably from Northern blots, we used TaqMan quantitative RT-PCR to assay RNA abundance in mg^L mutant brains. Comparing mg^L to control animals shows non-significant reduction in abundance of 5′ sequences (Figure 1C

), but ~6-fold loss of full-length transcript, represented by an assay 3′ to the mg^L insertion (Figure 1D

). However, this assay shows no effect of Nxf1 genotype on Atrn expression.

Figure 1

Nxf1^CAST does not suppress mRNA deficit in Atrn^mgL.

In contrast, for Mgrn, Nxf1-dependent differences in the level of correctly and alternatively spliced RNA isoforms from md alleles were readily quantified (Figure 2

). A probe 5′ to the md insertion (Figure 2A

) detects both normal and mutant-specific Mgrn RNAs (Figure 2B

). Correctly processed normal RNA is elevated in the presence of Nxf1^CAST, while levels of several mutant-specific transcripts is decreased (Figure 2B–D

), consistent with the mode of suppression previously reported for Pitpn^vb and Eya1^BOR. A probe 3′ to the insertion detects only the correctly spliced form, at levels comparable to the 5′ probe (not shown). Quantitative RT-PCR across the inserted intron confirms a ~2-fold increase in correctly-spliced transcript levels by Nxf1^CAST (Figure 2E

Figure 2

Nxf1^CAST suppresses pre-mRNA processing defects in Mgrn^md.

Nxf1^CAST Suppresses RNA, Protein, and Phenotypic Expression of Ap3d1^mh2J

To test Nxf1^CAST activity on a mutation for which protein level and phenotype were accessible, we analyzed RNA and protein levels, coat color (eumelanin) dilution and tremor severity of Ap3d1^mh2J mutant animals (Figure 3

). Locations of the mh^2J insertion and probes are indicated in Figure 3A

. Although Northern blots show high variance between experiments, comparisons between paired subjects examined on each blot shows a statistically significant increase in normal-sized Ap3d1 transcript and a modest decrease in mutant-specific transcript in the presence of Nxf1^CAST (Figure 3B–D

). Quantitative RT-PCR confirms the increase in correctly spliced RNA (Figure 3E

). Western blots show a corresponding increase in full-length Ap3d1 protein levels detected by an antibody to N-terminal residues (Figure 3F,G

). Correspondingly, a smaller protein species detected only in mutant animals is decreased in Nxf1^CAST animals. As predicted from this molecular analysis, Ap3d1^mh2J mutant animals also had improved pigmentation and neurological assessment scores in the presence of Nxf1^CAST as rated by observers blinded to genotype (Figure 3H–J

Figure 3

Nxf1^CAST suppresses Ap3d1^mh2J RNA, protein and visible phenotypes.

Nxf1^CAST Suppresses RNA, Protein, and Phenotypic Expression of Usp14^axJ

We similarly tested Nxf1^CAST activity on molecular and visible phenotypes of Usp14^axJ (Figure 4

). The insertion and probes used are indicated in Figure 4A

. Quantification of Northern blots and RT-PCR experiments from brain RNA shows significantly increased levels of correctly processed RNA in the presence of Nxf1^CAST (Figure 4B–D

). Quantification of Western blots shows that this is translated into an increased level of Usp14 protein (Figure 4E,F

). Behaviorally, Usp14^axJ mutant animals also showed improved neurological assessment scores, with visibly reduced tremor amplitude in the presence of Nxf1^CAST (Figure 4G

and Videos S1 and S2). In contrast to other mutations suppressed by Nxf1^CAST, normalized levels of mutant-specific isoforms of Usp14 RNA did not differ significantly by Nxf1 genotype. Comparing Northern blots hybridized with either 5′ or 3′ probes (as indicated in Figures 2

–

5),5

), we find Usp14^axJ and Eya1^BOR differ from other suppressed mutations in producing RNA isoforms that contain 5′ exons, IAP sequences and 3′ exons [4],[28] where most others produce primarily 5′ exons and terminal IAP sequences.

Figure 4

Nxf1^CAST suppresses Usp14^axJ expression and behavioral phenotypes.

Figure 5

Nxf1^CAST suppresses Atcay^hes RNA, protein and behavioral phenotypes.

Nxf1^CAST Suppression of Atcay^hes Indicates High Sensitivity of Phenotype to Increased Expression

To test Nxf1^CAST activity in the context of a human disease model, we analyzed several levels of molecular and behavioral phenotypes for the Atcay^hes mutation (Figure 5

). The locations of the hes insertion and probes are indicated in Figure 5A

. Atcay^hes alleles express prominent mutant-specific Atcay RNAs and very low levels of correctly processed full-length RNA [29]. Northern blots to quantify size-specific RNA levels show reduced level of each mutant-specific RNA detected by a probe 5′ to the insertion (Figure 5B,C

). A probe 3′ to the insertion detects only the full length “normal” RNA and is quantifiable only in non-mutant samples (not shown). To measure levels of normal RNA in mutant samples, we used a quantitative RT-PCR (TaqMan) assay (Figure 5D

). The presence of Nxf1^CAST significantly increases the level of correctly processed Atcay RNA accumulating from hes alleles. This difference is also translated into higher levels of the encoded Caytaxin/BNIP-H protein (Figure 5E,F

). Atcay^hes mutant animals have profound ataxia and an unusual jumping behavior (see Video S3). Nxf1 genotype had a highly significant impact on Atcay^hes neurological phenotypes as rated by multiple observers blinded to genotype, including both reduced ataxia and complete elimination of jumps from open field behavior (Figure 5G

and Videos S3 and S4).

Nxf1^CAST Does Not Suppress L1-LINE Mutation of Mitf^mi-bw

To test a non-viral class of retrotransposon, we examined whether Nxf1^CAST would suppress the black-eyed white L1-LINE insertion allele of Mitf. This mutation results in loss of pigmented melanocytes and extreme white spotting, leaving only occasional patches of pigment on the head or ears. Despite this low threshold for phenotype modulation, and known effects of other strain backgrounds, we saw no evidence for modification by Nxf1^CAST in an F2 cross. Among 14 Mitf^mi-bw, Nxf1^B6 and 9 Mitf^mi-bw, Nxf1^CAST doubly homozygous progeny, we observed a single animal of each genotype with dark patches on the head or ears.

Nxf1^CAST Does Not Suppress Typical ETn Insertions

We tested Nxf1^CAST activity on both sense and antisense-oriented ETn insertions of recent origin in both BALB/cJ and A/J mice. Expression levels of Zhx2 and its repression target Afp were assayed by quantitative RT-PCR from adult liver at P40 from 24 BALB/cJ x B6-Nxf1^CAST F2 animals selected by genotype (Figure 6A,B

). The BALB/cJ-derived insertion allele expressed ~1.5% non-mutant levels of Zhx2, with no difference between Nxf1 alleles. Similarly, the effect on Afp persistence, potentially a more sensitive indicator of Zhx2 function, showed no significant difference between Nxf1 alleles, although inter-individual variation was high (Figure 6B

, right panel), likely due to other factors segregating in this cross [39].

Figure 6

Nxf1^CAST does not suppress ETn-induced Zhx2^Afr mutation.

We tested the ability of Nxf1 to elevate transcript levels for another 5 sense and 3 antisense intronic ETn insertions in a second cross, A/J x B6-Nxf1^CAST (Figure 7

). Genomic organization and the location and orientation of the insertions are indicated (Figure 7A

). Quantitative RT-PCR measurements from brain or muscle (depending on known pattern of expression for each gene) showed no significant differences between Nxf1 genotypes for either sense or antisense insertions (Figure 7B,C

). A fifth sense-oriented insertion, in Prkca, showed no difference between inserted and uninserted alleles for either RNA or protein levels in this cross.

Figure 7

Nxf1^CAST does not suppress ETn-induced mutations in A/J.

Nxf1^CAST-Sensitive Insertions Carry the IΔ1 Deletion

Among sense-oriented IAP elements, only Atrn^mgL was not suppressed by Nxf1^CAST; as the inserted intron does not appear to be differentiated in position, length, or sequence composition from mutations that were suppressed (Figures 1

–

and data not shown) we determined the DNA sequence of each of these inserted elements, as well as the original Pitpn^vb insertion [4],[40]. We amplified each insertion using high-fidelity PCR optimized for long sequences, using unique primers flanking each insertion site (Supplemental material online). Ap3d1^mh2J, Atcay^hes, Mgrn1^md, Pitpn^vb and Usp14^axJ insertions all amplified fragments of 5.5 to 6.0 kb, while the Atrn^mgL insertion required modified conditions to support adequate amplification of a unique ~8 kb product. DNA sequence analysis showed that the Atrn^mgL element is a full length (type I) IAP, while each of Nxf1^CAST-sensitive elements includes the 1.9 kb deletion of gag-pol sequence typical of type IΔ1 elements [38] (Figure 8A

). All 6 elements belong to the IAPEz subfamily (www.repeatmasker.org), and contain an RTE-D transport element [41],[42] near the 3′ LTR. Calculated trees for each segment of aligned sequence shows that the full length Atrn^mgL element is not otherwise an outlier in overall sequence composition, except for the undeleted region of the gag gene (Figure 8B

). Inclusion in the tree of two recently identified IAP-IΔ1 insertions, Atp2b2^jog and Gria4^spkw1 [43],[44], suggests that they too should be sensitive to Nxf1^CAST-mediated suppression as they fall within sequence clades of suppressed elements for each segment.

Figure 8

The gag region and IΔ1 deletion differentiate Atrn^mgL from other IAP insertions.

Discussion

Our results show that Nxf1^CAST suppresses a broad and frequent class of IAP-induced mutations. The magnitude of increased normal transcript is ~2-fold and the impact on gene expression and behavioral phenotypes are significant in each case of this class examined. Nxf1^CAST increases the steady-state level of correctly spliced host gene transcript and almost always decreases the level of mutant-specific alternatively spliced transcript for six of seven sense-oriented IAP insertions examined to date (Table 1). The one exception, Atrn^mgL, differs from all of the suppressed elements we sequenced in having an intact gag-prt-pol coding sequence. Sequences within the deleted region may therefore mediate an additional level of Atrn repression that is not relieved by Nxf1^CAST. Each insertion, including Atrn^mgL also had a number of more subtle sequence variations, including smaller indels and further studies will be required to clarify which sequence differences contribute to the lack of suppression. However, the current data do provide a clear guide for the class of insertional mutation most likely to be quantitatively modulated by Nxf1^CAST, type IΔ1 IAPs, which are by far the most frequent class recovered from spontaneous mouse mutations. While it is possible that other genes within the congenic interval contribute to any one effect, transgenic mouse and lentiviral gene transfer studies with Pitpn^vb indicate that the main effect is due to Nxf1, as do the consistency of findings across all six suppressed mutations. Negative data from six ETn-inserted loci indicate that Nxf1^CAST is highly selective, and therefore unlikely to result in collateral changes in gene expression when used to manipulate IAP-induced mutations. Indeed, preliminary microarray data failed to identify any significant expression changes in whole brain RNA (B.A.H., unpublished data).

Table 1

Summary of genetic crosses to test Nxf1^CAST modifier effects.

The simplest explanation for the molecular data from the six mutations suppressed by Nxf1^CAST would be for Nxf1 to participate in pre-mRNA processing prior to the completion of splicing. This could occur by recruitment of Nxf1 to the nascent transcript by sequences in the IAP (or proteins bound to them co-transcriptionally) and subsequent interactions between Nxf1 and other components of the mRNP. Under such a model, amino acid differences (S48P and E610G) between the allelic Nxf1 proteins would alter the balance of alternative splicing either directly through interactions with splicing machinery or indirectly through an effect on transcriptional elongation rate or preference for termination site in the insertion. An alternative explanation might be for the export activity of Nxf1 to drive the nascent RNP into a territory with different relative activities for splicing and degradation, but this seems more difficult to reconcile with simultaneously increased levels of the correctly spliced message and decreased levels of the mutant splice form in five of the six suppression events.

Nxf1 protein interacts with several factors that could influence alternative splicing, including U2AF35 [45], several SR proteins [7],[8],[46],[47], and components of the TREX complex [48],[49]. Nxf1 is also recruited to the class of retroviral RNA transport elements (RTE-D), found in the IAPs we sequenced from suppressed mutations, through its interaction with RBM15 (OTT1) [42], which has also been linked to both splicing and export of Epstein-Barr virus mRNA [50]. Although these interactions are generally interpreted as recruiting export factors to mature RNPs [51], recruitment of Nxf1 to the nascent transcript through retroviral or cellular RNA transport elements could, in principle, alter the recruitment or activity of splicing factors. Both the RNA binding activity and much of the known protein interaction network around Nxf1 are conserved with respect to the Saccharomyces homolog, Mex67p [11],[48],[52]. It is interesting in this context that in splicing-specific RNA profiling of yeast mutations with defects in mRNA production the expression profile of MEX67-deficient strains cluster with transcriptional elongation factors [53]. Altered elongation rate is thought to be one mechanism that can regulate alternative splicing [54] and recruitment of Nxf1 to elongating nascent transcript could in principle alter the assembly or kinetics of other factors on the elongating pre-mRNA.

The extension of suppressor activity to a wider class of insertional mutations has several practical implications. First, these results predict that Nxf1^CAST should be able to modify other mutations that involve similar IAP insertions, for which new examples continue to be reported [43],[44],[55]. Indeed, the recent description of an IAP allele of Pofut1 notes variable reduction of phenotype among F2 progeny in a cross to CAST/Ei, the strain from which the suppressing allele of Nxf1 was derived [55]. The congenic Nxf1^CAST stock we have developed should be a useful tool to allow in situ titration of gene expression from either spontaneous or engineered alleles involving such insertions. Second, the range of titration in each of the six cases we have examined is ~1.5 to 2-fold and semi-dominant. This holds over a fairly broad range of mutational effects on gene expression, ranging from ~2% and 4% of wild-type levels (unsuppressed and suppressed, respectively) for Atcay^hes to 50% and 75% for Eya1^BOR. Finally, our in vivo gene titration results across six different mutations suggests that for a wide range of loci and allele strengths, even modest recovery of function may have dramatic phenotypic benefits. This is strikingly true in the case of Atcay^hes, where even a 2% increment of expression has a dramatic impact on behavioral phenotype (Videos S3 and S4). This implies that for Cayman ataxia, even a small amount of recovery in biochemical or cellular function would have substantial therapeutic benefit.

We have now demonstrated suppressor activity of the Nxf1^CAST allele toward six different mutations with distinct biochemical and physiological properties in the mouse. To the best of our knowledge this is now the most broadly validated suppressor or modifier gene activity in this well-studied species.

Materials and Methods

Mice

Congenic C57BL/6J (B6)–Nxf1^CAST mice were derived in our laboratory [4] and maintained by backcrossing to B6. Crosses described here were initiated with a stock at N19 or later backcross generation. C3H/HeJ–Atrn^mgL and B6–Mgrn^md were obtained from Dr. Teresa Gunn, Cornell University; mixed stock–Ap3d1^mh2J and C3H–Atcay^hes from Dr. Margit Burmeister, University of Michigan; B6–Usp14^axJ from Dr. Scott Wilson, University of Alabama, Birmingham; and B6–Mitf^mi-bw from Dr. Lynn Lamoreux, Texas A&M University. A/J and BALB/cJ were purchased from the Jackson Laboratory. Mice were maintained in specific pathogen-free conditions in accordance with protocols approved by the University of California at San Diego IACUC. Phenotypic comparisons were carried out using littermate pairs. Scores for behavioral phenotypes were assessed by at least 3 trained observers blinded to genotype. Videos of representative behaviors are available online as supporting information.

DNA

Genotypes for Nxf1 and each insertional mutation were determined by custom PCR assays for each locus. Conditions for PCR of full-length insertions were optimized using a commercial kit (MasterAmp Extra-Long PCR Kit, Epicentre) and primers in unique flanking sequences. DNA sequence analysis from the resulting PCR products used standard methods, as previously implemented in our laboratory [56] and assembled in Sequencher 4.8. Primers and PCR conditions are provided in the supporting information. Sequence alignments and neighbor-joining trees were performed in MUSCLE [57],[58] on the European Bioinformatics Institute web site (http://www.ebi.ac.uk/).

RNA

Freshly dissected tissues were homogenized in Trizol reagent (Invitrogen) and processed for RNA according to the manufacturers instructions. Poly(A)⁺ RNA was purified by oligo(dT) cellulose chromatography. Northern blots were prepared from formaldehyde-agarose gels by capillary transfer to Hybond-N membranes and crosslinked by exposure to 2400 J UV light. Probes were prepared from cDNA fragments by random primer labeling. Hybridizations to each filter were quantified by phosphorimage analysis (Storm, Molecular Dynamics) and normalized to subsequent hybridization of Gapd to the same membrane as an internal control. Quantitative PCR assays were performed on total RNA. TaqMan assays for Atrn (Applied Biosystems, assays Mm00437738_m1 and Mm01270975_m1) and Atcay (Mm01172843_m1) were performed by the UCSD Center for AIDS Research Genomics Core Laboratory and normalized to a Gapd TaqMan assay. All other quantitative RT-PCR experiments were performed using intron-spanning primers that flank the inserted intron, detected by SYBR green fluorescence in a Bio-Rad CFX96 instrument, and quantified by the ΔΔCt method. Measurements were performed in triplicate for each sample. Samples to be compared were measured on the same plate during a single run. Custom primer sequences and conditions are provided as Tables S1, S2, S3, and S4 online.

Protein

Freshly dissected tissues were homogenized in CelLytic M Cell Lysis reagent (Sigma #C2978) plus protease inhibitors and quantified using a bichromate assay (BCA, Pierce). Samples were subjected to SDS-PAGE and Western blotting onto Hybond-ECL membranes. Antibodies and dilutions used were goat anti-Ap3d1 (Rockland, 1 [ratio]

1000), rabbit anti-Caytaxin/BNIP (Gift of Dr. Low Boon Chuan [59], 1 [ratio]

5000), rabbit anti-Usp14 (Bethyl Laboratories, 1 [ratio]

5000). Relative levels of immunoreactivity were quantified using infrared dye-coupled secondary antibodies (Rockland, 1 [ratio]

10,000) on a LI-COR imager and normalized to rabbit anti-PITPβ [40] as an internal control that correlated with BCA-measured total protein.

Statistics

Summary data are plotted in figures as mean values, with error bars indicating standard deviations. For variables with expected normal distributions, including quantitative PCR experiments and behavioral observations in which several observers rated performance against a calibrated scale, hypotheses were tested using paired or unpaired t-tests depending upon whether the underlying materials were from explicitly paired samples (e.g., matched littermates) or aggregates (e.g., sibs and cousins). For variables expected to have non-normal distributions across trials (including blotting procedures, in which normalization and scaling across experiments complicate the analysis, and paired samples for which some replicate pairs represent different ages or breeding designs) hypotheses were tested using a nonparametric Wilcoxon signed-ranks test applied to replicates of paired experimental measures. Statistical calculations were carried out in Microsoft Excel or SISA online, http://www.quantitativeskills.com/sisa/ [60] (t-tests) or using the VassarStats public web interface, http://faculty.vassar.edu/lowry/VassarStats.html (Wilcoxon tests).

Supporting Information

Table S1

Genotype assays.

(0.02 MB XLS)

Click here for additional data file.^{(17K, xls)}

Table S2

qPCR assays.

(0.02 MB XLS)

Click here for additional data file.^{(17K, xls)}

Table S3

IAP insertions.

(0.01 MB XLS)

Click here for additional data file.^{(15K, xls)}

Table S4

Long PCR conditions.

(0.01 MB XLS)

Click here for additional data file.^{(14K, xls)}

Video S1

Video of behavioral phenotypes for Usp14axJ with Nxf1B6.

(0.26 MB MOV)

Click here for additional data file.^{(253K, mov)}

Video S2

Video of behavioral phenotypes for Usp14axJ with Nxf1CAST.

(0.58 MB MOV)

Click here for additional data file.^{(566K, mov)}

Video S3

Video of behavioral phenotypes for Atcayhes with Nxf1B6.

(1.20 MB MOV)

Click here for additional data file.^{(1.1M, mov)}

Video S4

Video of behavioral phenotypes for Atcayhes with Nxf1CAST.

(0.51 MB MOV)

Click here for additional data file.^{(501K, mov)}

Acknowledgments

We gratefully acknowledge Drs. Margit Burmeister, Teresa Gunn, Scott Wilson, and Lynn Lamoreux for providing mutant mouse stocks; Dr. Low Boon Chuan for anti-BNIP-H (Caytaxin) antibody; Wendy Alcaraz, Edward Chen, Peter Gent, Chen-Jie Hong, Anchang Hu, Eunnie Kim, Tiffany Poon, and Eric Raponi for assistance in scoring behavioral phenotypes; Pinyi Du for performing TaqMan assays in the UCSD Center for AIDS Research Genomics Core Laboratory; and Dr. Xiang-Dong Fu for helpful discussions and comments on a draft manuscript.

Footnotes

The authors have declared that no competing interests exist.

This work was supported by grant R01 MH59207 from the National Institute of Mental Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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