Integrator complex subunit 15 controls mRNA splicing and is critical for eye development

doi:10.21203/rs.3.rs-1748740/v1

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Integrator complex subunit 15 controls mRNA splicing and is critical for eye development

https://doi.org/10.21203/rs.3.rs-1748740/v1

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The eye and brain are composed of elaborately organized tissues, development of which is supported by spatiotemporally precise expression of a number of transcription factors and developmental regulators. Here we report the molecular and genetic characterization of Integrator complex subunit 15 (INTS15). INTS15 was identified in search for the causative gene(s) for an autosomal-dominant eye disease with variable individual manifestation found in a large pedigree. While homozygous INTS15 knockout mice are embryonic lethal, mutant mice lacking a small C-terminal region of INTS15 show ocular malformations similar to the human patients. INTS15 is highly expressed in the eye and brain during embryogenesis and stably interacts with the Integrator complex to support snRNA 3′ end processing. Its knockdown resulted in missplicing of a large number of genes, probably as a secondary consequence, and substantially affected genes associated with eye and brain development. Moreover, studies using human iPS-derived neural progenitor cells showed that INTS15 is critical for axonal outgrowth in retinal ganglion cells. This study suggests a new link between general transcription machinery and a highly specific hereditary disease.

The eye and brain are composed of elaborately organized tissues, development of which is supported by spatiotemporally precise expression of a number of transcription factors and developmental regulators. In addition, recent studies have pointed to the importance of alternative splicing. A large number of genes undergo dynamic changes in alternative splicing during neurogenesis and brain development^1–4. Defects in this intricate gene expression network are often associated with congenital anomalies in the eye and brain; more than 200 genes have been found associated with inherited retinal dystrophies alone ^{5, 6}. For example, mutations in PAX6, the master control gene for eye development that is expressed in numerous eye tissues in early stages of human development^{7, 8}, have been detected in various ocular anomalies, including aniridia, Peters anomaly, corneal dystrophy, and foveal hypoplasia, with some mutations found in the alternative exon 5a^9–11. With the advancement of technologies such as genome-wise association studies and next-generation sequencing (NGS), many more genetic defects will be found in brain- or eye-related diseases and conditions in the future.

Alternative splicing helps maintain the complexity of the transcriptome in higher organisms¹², and is performed by a multimeric ribonucleoprotein complex known as the spliceosome and nonspliceosomal RNA binding proteins¹³. Small nuclear RNAs (snRNAs), major components of the spliceosome, are processed by the Integrator complex¹⁴. Integrator was first discovered as a metazoan-specific 12-subunit protein complex that binds to the RNA polymerase II C-terminal domain and mediates co-transcriptional 3′ end processing of snRNA genes^14–17. Subsequently, two additional members of the complex were discovered, and Integrator is generally considered to have 14 subunits, INTS1–14. Thus, impairment of Integrator function leads to immature snRNA production and can result in secondary defects in mRNA splicing. Moreover, recent studies have shown that Integrator also regulates transcription of protein-coding genes and enhancer functions^18–20. At the organismal level, biallelic mutations of human INTS1 and INTS8 were reported to cause a neurodevelopmental syndrome^{21, 22}. In addition, INTS6 was found to be deleted in lung and esophageal cancers^{23, 24}.

Here we report the molecular and genetic characterization of INTS15, a putative 15th subunit of Integrator. INTS15, also known as C7orf26, has been identified as an interactor of Integrator in previous proteomic studies^25–27; however, its exact function remains unknown. Here we identified INTS15 in search for the causative gene(s) for a novel hereditary eye disease with an extremely unusual characteristic. Concordantly, mutant mice lacking a small C-terminal region of INTS15 showed ocular malformations similar to the human patients. Our molecular analysis revealed that the gene product stably interacts with the Integrator complex to regulate snRNA 3′ end processing. INTS15 is essential for survival at the cellular and organismal levels and plays an important role in mRNA splicing and genes associated with eye and brain development. Moreover, studies using human iPS-derived neural progenitor cells showed that INTS15 is critical for axonal outgrowth in retinal ganglion cells (RGCs). These findings illustrate a new link between general transcription machinery and a highly specific hereditary disease.

MOMS, an unusual human eye disease

We identified a large pedigree with a novel autosomal dominant developmental ocular anomaly, which we tentatively called multiple ocular malformation sequence (MOMS). Clinical examination revealed different combinations of various eye malformations, including corneal opacity, microcornea, iris anomaly, cataract, persistent fetal vasculature (PFV), foveal hypoplasia, myelinated retinal nerve fiber, atrophy of the retina and choroid, and a variety of optic nerve anomalies (Fig. 1a, b, Tab. 1, Extended Data Fig. 1). The anomalies in the pedigree were distributed throughout eye tissues. Furthermore, while hereditary eye diseases generally lead to similar phenotypes among affected family members and similarly affect both eyes of each patient²⁸, affected members of the MOMS family show a variety of anomalies in a variety of eye tissues that are incoherently combined in each eye. Such a phenotypic manifestation of MOMS is clinically unusual and has not been reported so far. Cataract surgery was performed in IV-2 and IV-4. Findings of eye anomalies have not changed during the follow-up period from childhood to adulthood in each patient, except for slight areal expansion of atrophy in the retina and choroid. No systemic or mental abnormalities were identified in any of the patients.

To identify the causative gene(s) for MOMS, we ran whole exome sequencing of three affected individuals (IV-2, IV-3, and IV-4). NGS revealed that 37 rare heterozygous single nucleotide variants (SNVs) were shared by the three patients (Fig. 1, Supplementary Data Set 1; see Methods for criteria for selecting candidate SNVs). These SNVs included four SNVs that affected protein sequences (Supplementary Data Set 1, Extended Data Tab. 1). Of the four SNVs, those in CD8B and FLOT2 were scored as polymorphism/benign by most tested programs, while SNVs in INTS15 and KBTBD8 were scored as disease causing and possibly_/probably_damaging by MutationTester and Polyphen2^{29, 30}. INTS15 is a ubiquitously expressed gene, whereas the other three genes are barely expressed in the central nervous system (Extended Data Tab. 1). The probability of being loss-of-function intolerant, a measure of gene essentiality ³¹, was high for INTS15, moderate for CD8B, and low for KBTBD8 and FLOT2. Incidentally, the Wnt signaling pathway is critical for retinal vascular development, its mutations being associated with PFV³²; however, the exome sequencing did not identify any SNVs that appear to affect the Wnt signaling pathway. Sanger sequencing confirmed the presence of the INTS15 c.C1274T, p.A425V variant in all seven patients examined (IV-2, IV-3, IV-4, III-1, III-2, and II-1) and its absence in two unaffected family members (IV-1, IV-5, and IV-6). Array-based comparative genomic hybridization (CGH) did not identify pathogenic copy number variations (CNVs) in the IV-2, IV-3, and IV-4 patients. From these results, we decided to focus on INTS15 for further functional investigation.

INTS15 knockout mice show phenotypes similar to human MOMS

We next generated INTS15-deficient mice using clustered regularly interspaced short palindromic repeat sequences (CRISPR), by targeting exon 6 or exon 2 of E130309D02Rik, the mouse ortholog of INTS15 (Supplementary Data Set 2). The exon 6 disruption, which is considered analogous to the INTS15exon 6 missense mutation found in MOMS patients, was performed by using catalytically inactive Cas9 fused to FokI nuclease and two sgRNAs to reduce off-target modification³³. Because exon 6 corresponds to the last exon, we also generated INTS15-deficient mice targeting exon 2 in order to fully eliminate its function. While exon 6-targeted mice were born in accordance with Mendelian inheritance, homozygous mice targeting exon 2 did not survive until birth and were recovered at a lower-than-expected Mendelian ratio at the blastocyst stage (Fig. 2A), suggesting that INTS15 is essential for early embryonic development.

PFV, persistence of ocular fetal vasculature that normally regresses by apoptosis during eye development, is the most common feature in MOMS patients. Compared to wild type mice (Fig. 2B), heterozygous INTS15 ^{exon 6 +/-} mice showed PFV on the optic nerve head (Fig. 2C). Homozygous INTS15 ^{exon 6 -/-} mice had more severe ocular phenotypes in various eye tissues than its heterozygous littermates (Tab. 2). In the homozygous mice, PFV was also common and tend to be more prominent, extending from the optic nerve head to the posterior surface of the lens and occupying a large area in the vitreous cavity. Ophthalmoscopy identified local chorioretinal atrophy, which was histopathologically identified as defect of the choroid, deformity of retinal layers, and gliosis (Fig. 2D). Corneal opacity, cataract, and optic nerve anomaly were also seen, but at low incidence (Tab. 2). All of these phenotypes were similar to those of MOMS patients.

Heterozygous INTS15 ^{exon 2 +/-} mice carrying a 7-bp deletion in exon 2 exhibited even more severe malformations throughout eye tissues than homozygous INTS15 ^{exon 6 -/-} mice (Fig. 2E, Tab. 2), some of which were never seen in MOMS patients. Prominent PFV, chorioretinal atrophy, corneal opacity, cataract, and optic nerve anomaly, which were characteristic of MOMS patients, were more frequently seen in INTS15 ^{exon 2 +/-} mice than in INTS15 ^{exon 6 -/-} mice. On the other hand, anterior segment dysgenesis with adhesion of the cornea and iris, ectopic lens, retinal folding, and microphthalmos with whole eye tissue dysgenesis were not seen in INTS15 ^{exon 6 -/-} mice and MOM patients. Thus, phenotypic manifestations of INTS15 ^{exon 2 +/-} mice were similar to, but more diverse than, those of the human patients, suggesting that haploinsufficiency of INTS15 has a broader impact on eye development. These findings support the idea that INTS15 is a causative gene for MOMS.

INTS15 expression patterns and its role in cell survival

We explored INTS15expression patterns during development and in adult mice. In situ hybridization in wild-type mouse embryos at E8.0 showed that INTS15is predominantly expressed in the brain and eyes (Fig. 3A). Immunohistochemical analyses in wild type mouse embryonic cells also showed predominant expression of INTS15 in the nuclei of brain and eye primordial cells, with ubiquitous expression in the rest of the body and human cell lines (Fig. 3B). The mRNA expression profile of INTS15 in the Human Protein Atlas showed that it is ubiquitously expressed throughout the body.

To further assess the role of INTS15in cell survival, we performed knockdown experiments using two different approaches. First, we transfected pU6-shINTS15-GFP for strong and stable reduction in HeLa cells (Extended Data Fig. 2b), in which endogenous INTS15 is expressed in the nucleus, and analyzed for the presence of an apoptotic marker, M30³⁴. M30 and diffuse DAPI staining characteristic of apoptotic cells were observed 36 h after transfection of the INTS15 knockdown vector (Fig. 3C). The induction of apoptosis was rescued by simultaneous transfection of a INTS15-Flag expression vector (Fig. 3D), suggesting that INTS15 is required for cell survival.

INTS15 functions together with Integrator in snRNA 3′ end processing

Although INTS15 encodes a 449 amino acid protein of unknown function, three independent, high-throughput mass spectrometry analyses have consistently shown that INTS15 coimmunoprecipitates with some subunits of the Integrator complex^25-27. Concordantly, molecular phylogenetic analysis showed that INTS15 is conserved in vertebrates and Drosophila, similarly to the other established Integrator subunits (Extended Data Fig. 3). We therefore carried out coimmunoprecipitation analysis using 293T cells overexpressing INTS15-Flag or INTS5-Flag. INTS4, INTS5, INTS10, and INTS13 were coimmunoprecipitated with anti-Flag beads from cells overexpressing INTS15-Flag, but not from cells overexpressing control GFP (Extended Data Fig. 4a-e). INTS15 was coimmunoprecipitated with anti-Flag beads from cells overexpressing INTS5-Flag (Fig. 4a). Moreover, anti-INTS5 antibody coimmunoprecipitated endogenous INTS15 along with INTS5 and several other INTSs from HeLa cell nuclear extract (Fig. 4b). Blue native polyacrylamide gel electrophoresis of mouse adult brain extract also showed that endogenous INTS15 was comigrated with INTS4, INTS10, and INTS11 (Extended Data Fig. 4f). Overall, these results are consistent with the previous proteomic studies and suggest that INTS15 stably interacts with the Integrator complex. INTS15 has no known conserved domains or motifs. To gain structural insight into INTS15, we tested a series of deletion mutants of INTS15 for Integrator binding. None of the mutants coimmunoprecipitated INTS5 appreciably (Extended Data Fig. 5), suggesting that the entire sequence of INTS15 is required for its binding to Integrator.

Since Integrator plays a pivotal role in the 3′ end processing of RNAPII-transcribed pre-snRNAs ^14-17, we investigated the effect of knockdown of INTS15 on this process. Quantitative (q) RT-PCR was performed to assess the amount of total snRNAs and unprocessed snRNAs. Compared to control HeLa cells, the amounts of unprocessed U1, U2, U4, U5A, and U11 snRNAs were significantly increased in HeLa cells transfected with INTS15 siRNA, whereas the total amounts of snRNAs were relatively unaffected by INTS15 knockdown (Fig. 4c, Extended Data Fig. 2a). Thus, like Integrator, INTS15 is critical for snRNA 3′ end processing.

INTS15 regulates genes associated with brain and eye development

To investigate possible consequences of INTS15 knockdown on gene expression, we performed microarray analysis. We identified 700 transcripts that were downregulated by 50% or more in INTS15 siRNA-treated HeLa cells (Supplementary Data Set 3). Gene set enrichment analysis showed that genes tagged with ‘alternative splicing’ or ‘splice variant’ (i.e., genes from which at least two splice variants are generated) occupy a large fraction of the downregulated genes (Fig. 4d). Thus, genes with splice variants appear to be major targets of INTS15. This finding led us to hypothesize that changes in mRNA splicing are caused by INTS15 knockdown through defects in snRNA 3′ end processing. To test this idea, we performed whole transcriptome sequencing of INTS15 siRNA-treated HeLa cells. To avoid rapid degradation of misspliced mRNA via nonsense-mediated mRNA decay, mRNA samples were obtained from HeLa cells treated with cycloheximide for 6 h prior to harvest. As expected, 2,777 missplicing events (with a threshold level of P < 0.01) were identified specifically in INTS15 siRNA-treated HeLa cells (Fig. 4e, Supplementary Data Set 4). Concordantly, RT-PCR analysis showed that INTS15 knockdown induces intron retention of F12 and exon skipping of GGA1 and IKBKG (Fig. 4f). Together, these results suggest that the knockdown of INTS15 results in missplicing of a large number of genes.

Considering the eye phenotypes of human MOMS patients and INTS15 knockout mice and its high-levelexpressionin the developing brain, we analyzed genes associated with these organs using the above RNA-seq data. Numerous transcripts associated with eye and brain development, including PAX6, the master control gene for eye development⁷, were found to be downregulated by INTS15knockdown (Fig. 4g, h, Supplementary Data Set 5, 6).

We previously generated brain-like and retinal tissues from human iPS cells³⁵. Using the procedure shown in Extended Data Fig. 6, brain-like and retinal tissues containing RGCs, with axons extending radially from the margin of the retinal tissue, are obtained at a high yield (Extended Data Fig. 6). RGC axons are major components of the optic nerve. Thus, we used this in vitro model to directly test the role of INTS15 in brain and eye development. iPS-derived retinal tissue containing RGCs developed abnormally, being unable to elongate axons, when INTS15 siRNA was transfected on Day 27 (3 days before analysis), when axons were not yet significantly elongated (Fig. 4i). Next, brain-like and retinal tissues of the organoids were respectively microdissected and subjected to qRT-PCR analysis (Extended Data Fig. 6). In the presence of INTS15siRNA, expression of the developing brain cell markers BF1 and FGF8 was significantly decreased in the brain-like tissue (Fig. 4j). Expression of the developing RGC markers BRN3B and ISLET1 was also significantly decreased inthe retinal tissue (Fig. 4k). These results suggest that INTS15 plays an important role in neural development in the eye and brain, possibly via regulation of mRNA splicing. Because retinal dysplasia and optic nerve hypoplasia are associated with malformation of RGCs and their axons^{36, 37}, the above results are consistent with the idea that functional defects of INTS15 in RGCs caused some of the phenotypes characteristic of MOMS.

Starting from genetic analysis of a novel hereditary eye disease, we presented evidence that INTS15 is indeed a novel subunit of the Integrator complex. Although previous proteomic studies have found that INTS15 coimmunoprecipitates with some Integrator subunits^25–27, little has been known about its molecular and physiological functions. INTS15 stably interacts with the Integrator complex and its transient knockdown using siRNA results in defects in snRNA 3′ end processing, the phenotypes observed when known Integrator subunits are knocked down. This study also clearly illustrated that INTS15 is critical for ocular development at the organismal level. Since this study is based on a small number of patients from a single large pedigree, it is premature to conclude that INTS15 is causative for MOMS. Detailed diagnostic and molecular studies could not be performed on the other members of the pedigree, and linkage analysis was not performed due to the small sample size. Given the phenotypic similarities between MOMS patients and INTS15 KO mice, however, we consider it likely that the missense mutation found in INTS15 contributes to the onset of MOMS.

While stable knockdown of INTS15 in HeLa cells using vector system resulted in apoptosis, its transient knockdown using siRNA not only resulted in defects in snRNA 3′ end processing, but also affected splicing of a subset of protein-coding genes and in vitro generation of eye and brain tissues from human iPS cells. Concordantly, while homozygous INTS15 knockout mice were embryonic lethal, its haploinsufficiency or deletion of a small C-terminal region, the latter of which being more similar to the A425V mutation found in MOMS, resulted in variable ocular malformations similar to the human patients. Incidentally, targeted disruption of mouse Ints1 causes growth arrest at the early blastocyst stage and eventual apoptotic cell death³⁸. Moreover, multiple biallelic, missense or truncating mutations in human INTS1 and INTS8 have been found to cause a distinct neurodevelopmental syndrome characterized by developmental delay, craniofacial anomalies, motor impairment, and cataract^{21, 22}. Thus, hypomorphic mutations in INTS15 and a canonical INTS seem to cause a spectrum of clinical manifestations with some commonality.

Although it is unclear exactly how an impairment of INTS15 function could cause the unique phenotypes of MOMS with a variety of multiple ocular developmental defects occurring in combination, a plausible explanation is that it causes defects in mRNA splicing or changes in alternative splicing by blocking the production of functional snRNAs, and that the resulting gene expression changes interfere with normal eye development. Indeed, alternative splicing has been shown to be particularly important for the development of the eye and brain, representative examples being the regulation of a neuron-specific transcription factor with multiple splice variants^{1–4, 11}. Since the eye and brain have intricate, highly organized structures, it seems likely that these organs are most sensitive to subtle mutations affecting transcription and mRNA splicing. Whatever the case, this study establishes a new link between general transcription machinery and a highly specific hereditary disease.

Ethics statement

All analyses performed in this manuscript were carried out in accordance with the Declaration of Helsinki and were approved by the Ethics Committee of the National Center for Child Health and Development, Tokyo, Japan (#518, genetic analysis; #686, experiments using human iPS cells). Written informed consent from the patients or their parents was obtained from all members. All animal protocols were approved by the Animal Care and Use Committee of the National Research Institute for Child Health and Development, Tokyo, Japan. Generation and all experiments of knockout mice were conducted in accordance with the approved animal protocols (#A2015-005-C03).

Subjects

Seven patients of the family visited our hospital and were interviewed about the eye and systemic symptoms of family members. Seven patients underwent comprehensive eye examinations, including slit lamp examination, ocular tonometry, fundus examination and visual acuity testing.Swept source optical coherence tomography was measured using DRI OCT Triton (TOPCON, Japan), and electroretinogram was measured using RETeval (Welch Allen, LKC Technologies, USA) in accordance with International Society of Clinical Electrophysiology of Vision (ISCEV) protocol. For genomic analysis of the seven patients, DNA samples were obtained from peripheral leukocytes. In addition, DNA samples were obtained from peripheral leukocytes or saliva of two unaffected family members (IV-1, and IV-6). Of these, three patients (IV-2, IV-3, and IV-4) were subjected to systematic molecular analyses using array-based CGH and NGS. The three patients (IV-2, IV-3, and IV-4), the remaining four patients (II-1, III-1, III-2, and IV-5), and two unaffected individuals (IV-1 and IV-6) underwent Sanger sequencing for the SNVs detected in IV-2, IV-3, and IV-4.

Molecular genetic analyses of MOMS

CNVs in the genome were analyzed by CGH using a catalog human array (4 × 180 k format; Agilent Technologies, Palo Alto, CA, USA). The Database of Genomic Variants³⁹ was used to exclude benign CNVs. SNVs were screened by NGS using the SureSelect All Exome v5 kit (Agilent Technologies). The library was sequenced as 100-bp paired-end reads using a HiSeq 2000 sequencer (Illumina, San Diego, CA, USA). Sequence data were analyzed by using Avadis NGS 1.3.1 (DNA Chip Research, Yokohama, Japan) and SAMtools 0.1.17 software⁴⁰. The population frequency of identified SNVs was assessed using dbSNP⁴¹, the 1000 Genomes Browser, and the Human Genetic Variation Browser⁴². Functional consequences of SNVs were predicted by using Polyphen2²⁹, SIFT, and MutationTaster2³⁰. SNVs with low signal quality scores were excluded from further analyses. Since MOMS appears to be an autosomal dominant disease with a relatively high penetrance, we searched for SNVs meeting the following criteria: (i) those present in a heterozygous state in all the three subjects examined; (ii) those accounting for less than 0.1% of the general population; and (iii) those absent in the control samples obtained from eight unaffected Japanese individuals. Then, we selected SNVs that affect protein sequence or a splice site. Tissue expression patterns of the affected genes were examined using the GTEx portal. The probability of being loss-of-function intolerant was assessed by using the data reported by Lek et al³¹. The results of NGS were validated by Sanger sequencing. Furthermore, we performed Sanger sequencing for seven individuals of this family, three affected individuals and four unaffected individuals, to examine whether the detected SNV co-segregated with MOMS.

Generation of E130309D02Rik exon 2 and exon 6 knockout mice

Plasmids containing human codon-optimized Cas9 (hCas9) and single-guide RNA (sgRNA) cloning vector were gifted from George Church (Addgene plasmid #41815 and #41824, respectively)⁴³. A plasmid containing inactive Cas9 (dCas9) fused with the FokI DNA nuclease domain (FokI-dCas9; fCas9) was a gift from David Liu (Addgene plasmid #42970). Specific sgRNA sequences were designed using CRISPRdirect⁴⁴. sgRNAs for exon 6 and exon 2 of INTS15, hCas9 mRNA, and FokI-dCas9 mRNA were prepared as previously described^{45, 46}. Three sgRNA target sequences were cloned into sgRNA cloning vectors by inverse PCR using primers pairs INTS15 exon 6_sg1-F / INTS15 exon 6_sg1-R, INTS15 exon 6_sg2-F / INTS15exon 6_sg2-R, and INTS15exon 2_sg-F / INTS15 exon 2_sg-R (Supplementary Data Set 2). In vitro transcription was performed using the mMESSAGE mMACHINE T7 Transcription Kit (Ambion, Austin, TX, USA). PCR products amplified using T7-added primers (T7-INTS15 exon 6_sg1 for INTS15 exon 6_sg1, T7- INTS15 exon 6_sg2 for INTS15 exon 6_sg2, and T7- INTS15 exon 2_sg for INTS15 exon 2_sg; Supplementary Data Set 2) were used as templates. Transcribed RNAs were purified using the MEGAclear RNA Kit (Ambion).

Superovulated F1 hybrid of C57BL/6 × DBA/2 (BDF1) female mice were crossed with BDF1 male mice. The fertilized mouse zygotes were collected in M2 medium. Microinjection was carried out as previously described⁴⁷. Essentially, RNAs and ssODNs were mixed just before microinjection into the cytoplasm or pro-nuclei of zygotes, and the injected embryos were incubated at 37ºC until they were transferred into pseudopregnant females at the two-cell stage. The concentration of injected RNAs was always kept at 500 ng/ml in total. For the sgRNA/hCas9 condition, sgRNA and hCas9 mRNA were mixed at a 1:1 ratio, and thus the final concentration was 250 ng/ml each, and for the two sgRNAs/FokI-dCas9 condition, the sgRNAs and FokI-dCas9 mRNA were mixed at a 1:1:1 ratio, and thus the final concentration was 167 ng/ml each. The final concentration of injected ssODNs was 100 ng/ml. All mice were obtained from Sankyo Labo Service (Tokyo, Japan).

Genomic DNA was extracted from the fingers of day 4 to 7 neonates using Kaneka Easy DNA Extraction Kit version 2 (Kaneka #KN-T 110005). For direct sequence analysis, the genomic regions around gRNA target sites were PCR amplified using the primers shown in Supplementary information Dataset S7. The obtained PCR products were treated by ExoSAP-IT Express PCR Product Cleanup Regent (Thermo Fisher Scientific #75001) and sequenced directly using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific #4337454). To confirm the sequence of the mutated alleles, PCR products were also analyzed by 2% (for Exon2F / Exon2R products) or 2.5% (for Exon6F / Exon6R products) agarose gel electrophoresis and were cloned using the TOPO TA Cloning Kit (Thermo Fischer Scientific #K4575J10). After PCR amplification of the insert using M13 forward and reverse primers, positive clones were subjected to sequence analysis using T7 forward and M13 reverse primers. To detect CRISPR/Cas9-mediated INTS15 exon 2 knockout, a 136-bp fragment including the gRNA target site was amplified from genomic DNA using the primer set INTS15 exon 2 forward and INTS15 exon 2 reverse indicated in Extended Data Tab. 1 and electrophoresed on Extra PAGE One Precast Gel (Nacalai Tesque #13076-14) to detect 7-bp deletion.

Mice at the age of 1 or 2 months were examined with slit-lamp biomicroscopy and ophthalmoscopy. When abnormalities were detected, mice were sacrificed by cervical dislocation, and organs including the eye, brain, heart, lung, intestine, and kidney were examined under a stereoscopic microscope.

Collection and genotyping of blastocysts

Four-week-old INTS15^{exon 2+/-} female mice were superovulated with 5 IU of pregnant mare serum gonadotropin (Folligon; Intervet, Bendigo East, Australia) administered intraperitoneally, followed 48 hours later by 5 IU of human chorionic gonadotropin (Chorulon, Intervet), and were mated with INTS15^{exon 2+/-} male mice of the same strain overnight. Mice were sacrificed by cervical dislocation, and preimplantation embryos were collected. Noon of the day when a vaginal plug was detected was designated embryonic day 0.5 (E0.5). E3.5 blastocysts were isolated by flushing uteri with M2 medium.Genotyping was performed with direct PCR. Individual blastocysts were separated on dishes containing M2 medium and transferred into tubes containing 1 μl of sterile water, to which 3 μl of lysis buffer (0.05% SDS and 0.035 N NaOH) was added. After incubation for 5 min at 95 °C, lysates were subjected to direct PCR in a total volume of 50 μl with LA Taq polymerase (TaKaRa Bio) using the primers described in Supplementary Data Set 7.

RNA in situ hybridization

Whole-mount in situ hybridization was performed as previously described⁴⁸. A gene-specific fragment of E130309D02Rik was amplified from mouse chondrocyte cDNA by PCR using the following primers: forward, 5′-TTGAGACCTTCGACGTCAATGAG-3′ and reverse, 5′-TCTGGCCACGAGTGGCC-3′.

Paraffin section preparation, hematoxylin and eosin staining and immunohistochemistry

After sacrificing mice by cervical dislocation, eyes were enucleated, fixed in SUPER FIX (KURABO #KY-500), rinsed in water, dehydrated in a graded series of alcohol, and embedded in paraffin, as described previously¹¹. Each block was serially sectioned at 3 μm in thickness. Deparaffinated sections were stained with hematoxylin and eosin. For the immunoenzyme method, avidin-biotin complex methods were performed using VECTASTAIN ABC kit (Vector laboratories #PK-6100) according to the instructions, and observed by using an IX71 inverted microscope (Olympus).

Immunofluorescence

Specimens were fixed with 4% paraformaldehyde (pH 7.0) for 20 min at room temperature and subjected to immunofluorescence staining as described previously³⁵. The antibodies used in this study and dilution rates are listed in Supplementary Data Set 8.

Plasmids

Human INTS15 (NM_024067) ORF clone with C-terminal Myc-DKK tag (Origene #RC219786; pCMV C7orf26-Flag) and negative control GFP expressing clones (TaKaRa Bio #Z2470N; pAcGFP1-C1) were purchased. INTS15 shRNA clone tagged with GFP (GeneCopoeia #HSH018872-CU6; pU6-shINTS15-GFP) and negative control shRNA clone (GeneCopoeia #CSHCTR001-CU6; pU6- non-specific shRNA-GFP) were also purchased. These expression vectors were prepared using EndoFree Plasmid Maxi Kit (QIAGEN #12362).

Cell culture

HeLa cells were obtained from ATCC, and 293T cells was purchased from TaKaRa Bio. HeLa cells were grown in DMEM medium (Thermo Fisher Scientific #11965092) supplemented with 10% FBS and 1% Pen Strep, whereas 293T cells were cultured in DMEM medium with 10% FBS, 1×GlutaMAX Supplement (Thermo Fisher Scientific #35050061), and 1% Pen Strep. Neonatal normal human dermal fibroblasts NHDF (Lonza #CC-2509) was purchased and cultured using Fibroblast Growth Medium-2 (Lonza #CC-3132). Human iPSCs (HPS0007_409B2 cell passage 29) were obtained from RIKEN BioResource Center (Tsukuba, Japan) and were maintained on Laminin-511 E8-coated 6-well plates in StemFit AK02N (Ajinomoto, Tokyo, Japan) for feeder-free culture. For passaging, hiPSCs were detached by treatment with TrypLE Express (Thermo Fisher Scientific) at 37°C for 8 min using a cell scraper. After collection by centrifugation, cells were suspended in medium containing 10 µM Y-276 and seeded at 10,000 cells per well.

Knockdown of INTS15 by siRNA and shRNA

siRNA specific for INTS15 (ON-TARGETplus Human INTS15 (79034) siRNA SMARTpool, #L-016351-02) and negative control siRNA (ON-TARGETplus Non-targeting Pool, #D-001810-10; NS siRNA) were purchased from Dharmacon. Seeded in 24-well plates 2 days before transfection, HeLa cells at a cell confluency of 50–70% were transfected with 50 µL of Opti-MEM I medium containing 5 pmol of siRNA using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific #13778150) or with 50 µL of Opti-MEM I medium containing 500 ng of pU6-shINTS15-GFP or pU6-non-specific shRNA-GFP using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific #L3000001) in accordance with the manufacturer’s instructions. Cells were harvested or fixed at different time points after transfection for subsequent analysis.

Apoptosis assay

HeLa cells were transfected with pU6-shINTS15-GFP or pU6-non-specific shRNA-GFP, cultured in 96-well plates or 8-well glass chambers, and fixed 36 h after transfection. After two washes with PBS containing 0.1% Tween 20, fixed cells were incubated with M30 CytoDEATH antibody solution (1:50) (Roche #12140322001) for 60 min at room temperature. Cells were washed twice and incubated with goat anti-mouse IgG (H+L) secondary antibody and Alexa Fluor Plus 555 solution (1:500) (Thermo Fisher Scientific #A32727) for 30 min at room temperature. After mounting with ProLongGold Antifade Reagent with DAPI (Thermo Fisher Scientific #A32727), M30-positive cells were observed with a DeltaVision Elite microscope (CORNES Technologies).

Immunoprecipitation and immunoblot analysis

293T cells at a confluency of 70–80% in 10 cm dishes were transfected with 1.5 mL of Opti-MEM I medium containing 15 µg of pINTS15-myc-flag using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific #L3000001). After 72 h culture, protein was extracted from the cells using CelLytic M Cell Lysis Reagent (Sigma #C2978) containing protease inhibitor cocktail (Roche #04693116001) in accordance with the manufacturer’s instructions. Immunoprecipitation was performed using EZview Red ANTI-FLAG M2 Affinity Gel (Sigma #F2426) and FLAG Immunoprecipitation Kit (Sigma #FLAGIPT1). Affinity gel was suspended with 1% BSA washing buffer and rotated for 30 min at room temperature. After four washes with washing buffer, the affinity gel was suspended with the cell lysate and rotated for 16 h at 4^oC. After four washes with washing buffer, Flag-tagged INTS15 was eluted from the affinity gel with 3×Flag peptide solution. After concentrating the volume using Amicon Ultra-0.5 (Millipore #UFC5003), protein amounts were determined using the Qubit Protein Assay (Thermo Fisher Scientific #Q33211). Approximately 5 μl of extracts and 1 μl of clarified eluates were boiled for 5 min at 95^oC, separated on an 7.5% acrylamide SDS gel, and transferred to PVDF membranes (Bio-Rad #1620174). Membranes were blocked with Blocking One (Nacalai Tesque #03953), incubated for 20 min at room temperature and washed with TBST. The membranes were incubated with primary antibodies against INTS4 and INTS5 (Supplementary Data Set 8) at 4^oC overnight, washed with TBST, and incubated for 1 h at room temperature with HRP-conjugated secondary antibodies (Supplementary Data Set 7)diluted in Blocking One. Immunoreactive bands were visualized with SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific #34095). For coimmunoprecipitation analysis of endogenous proteins, anti-INTS5 (Supplementary Data Set 8) was immobilized to protein A agarose beads and then incubated with nuclear extract prepared from HeLa cells. After extensive washing, bound material was eluted with Laemmli sample buffer for immunoblot analysis.

BN-PAGE

BN-PAGE was performed as described previously⁴⁹. Nuclear fraction was extracted from adult mouse brain by NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific #78833) according to the manufacturer's protocol. Approximately 20 µg solubilized proteins were then resolved on BN-PAGE (4–12%), which was followed by Western blot analysis. Molecular weights were determined using the NativeMark Unstained Protein Standard (Life Technologies).

RNA-seq analysis

Poly(A)+ mRNA fractions were isolated from HeLa/INTS15 siRNA cells and negative control HeLa/NS siRNA cells 48 or 72 h after transfection, and whole transcriptome analysis was performed by using HiSeq 2500 (Illumina) to obtain 20 million reads per sample. Cells were treated for 6 h before RNA extraction with 30 µg/mL cycloheximide (Sigma #C7698). Paired-end reads were mapped to the human genome GRCh38.p13. Differential splicing events between the two conditions were analyzed by using the software Multivariate Analysis of Transcript Splicing⁵⁰. Heatmaps were created using log2 (FPKM) of each transcript. Transcripts were categorized based on GO terms such as brain development and eye development. Transcripts with FPKM of less than 1.0 throughout the samples were excluded from further analysis.

Splicing assays

To confirm intron retention, exon skipping, and alternative splicing events in the transcripts obtained from HeLa/INTS15 siRNA cells and HeLa/NS siRNA cells, primers sets (Supplementary Data Set 7) were designed so that they amplify splice variants in question simultaneously, and semi-quantitative RT-PCR products were analyzed on agarose gels.

Knockdown of INTS15 in iPS-derived retinal and brain tissues

Human cortical and retinal tissues were induced from human iPS cells as described previously³⁵. iPS-derived embryoid bodies with optic vesicles, which are primordia of the brain and retina, were adhered to 24-well plates 22 days after the start of neuronal differentiation. After 48 h, culture medium was replaced with Accell siRNA delivery media (Dharmacon B-005000-100) containing 100 µM Accell Human siRNA (Dharmacon C7orf26, E-016351-00-0005; Non-targeting, D-001910-01) and then cultured for 3 days. Retinal and brain tissues were dissected from colonies for subsequent qRT-PCR analysis.

qRT-PCR analysis of INTS15 knockdown in HeLa cells

Total RNA was extracted from HeLa/INTS15 siRNA and HeLa/NS siRNA cells 48-72 h after transfection using RNeasy Mini Kit (QIAGEN #74136), and its concentration determined using a NanoDrop LITE (Thermo Fisher Scientific). Expression of mRNAs or snRNAs in each RNA sample was determined by two step qRT-PCR using StepOnePlus Real-Time PCR System (Applied Biosystems) as previously described³⁵. C_T values were normalized to the expression levels of HPRT1 for mRNA and 7SK for snRNA, and relative amounts of target RNAs were calculated using the 2^–ΔΔCT method. The primers used in this study are listed in Supplementary Data Set 7.

Molecular phylogenetic tree

Amino acid sequences of INTS15 of various species were obtained from the Ensembl database. The phylogenetic tree was constructed using the neighbor-joining method and ClustalX software. The reliability of the tree was estimated using the bootstrap method and 10,000 replications.

Microarray analysis

Total RNA extracted from siRNA-treated HeLa cells (50 ng) was used to prepare Cy3-labeled target cRNA with the Low Input Quick Amp Labeling Kit (Agilent Technologies). The resultant cRNA (600 ng) was hybridized to Sureprint G3 Human Gene Expression Microarray 8 × 60 K v3 (Agilent Technologies), and the signal intensities were scanned according to the manufacturer’s protocol. The data were analyzed using GeneSpring GX software (Agilent Technologies). 75 percentile normalization was chosen as the normalized method, and only the probes that were “detected” in at least one sample in the control/target cell samples were used for further analysis. The number of probes used in the analysis was 27,865. The differentially expressed genes were selected using the criteria of |log2 (fold change)| ≥ 1. The functional annotation chart for downregulated genes was obtained using DAVID v6.5⁵¹.

Statistics

Statistical analysis was performed using Microsoft Excel statistics software. All values are expressed as mean ± SD. Unpaired two-tailed t-tests were used to compare groups. P values less than 0.05 were considered statistically significant; individual P values are indicated by asterisks in graphs (*, P < 0.05).

Data availability

RNA-seq data are available at Gene Expression Omnibus (accession number: GSE186331, secure token: cnilkwcylvgnnyv). Microarray data are available at Gene Expression Omnibus (accession number: GSE186323, secure token: utoxwsgqxtynxcb).

Acknowledgements

We thank T. Gojobori for helpful discussions, M. Miyado for technical support, Y. Fukamachi and Y. Takahashi of Mitsui Knowledge Industry for advice for data mining, and H. Pickersgill of Life Science Editors for English editing. We thank Y. Uemura and K. Minoda, who introduced us to the patients and gave us an opportunity to investigate the INTS15 gene. This work was supported by the JSPS KAKENHI (grant number 17K19738), the AMED (grant number 17ek0109217h0001), and a grant from the National Center for Child Health and Development (number 28-2).

Author contributions

N.A. directed the project; Ta.Yo., Y. Y., and N.A. wrote the manuscript; Ta.Yo., S.N., and N.A. collected medical record; M.F., K.O., and K.M. performed exome analysis; Ta.Yo., E.M., M.T., S.T., and N.A. performed knockout mice experiments and collected data; E.M. and T.T. cultured cells and performed in vitro knockdown experiments and PCR analysis; Ta.Yo., To.Ya., T.T., J.H., Y.S., and Y.Y. performed immunoprecipitation and Western blotting; H.N. and H.H. contributed to interpretation of the results and provided advice on the experiments.

'Competing Interests' statement

The authors declare no competing financial or non-financial interests.

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Integrator complex subunit 15 controls mRNA splicing and is critical for eye development

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