Human Intron Research Articles

Role of U11/U12 minor spliceosome gene ZCRB1 in Ciliogenesis and WNT Signaling.

Despite the fact that 0.5% of human introns are processed by the U11/U12 minor spliceosome, the latter influences gene expression across multiple cellular processes. The ZCRB1 protein is a recently described core component of the U12 mono-snRNP minor spliceosome, but its functional significance to minor splicing, gene regulation, and biological signaling cascades is poorly understood. Using CRISPR-Cas9 and siRNA targeted knockout and knockdown strategies, we show that human cell lines with a partial reduction in ZCRB1 expression exhibit significant dysregulation of the splicing and expression of U12-type genes, primarily due to dysregulation of U12 mono-snRNA. RNA-Seq and targeted analyses of minor intron-containing genes indicate a downregulation in the expression of genes involved in ciliogenesis, and consequentially an upregulation in WNT signaling. Additionally, zcrb1 CRISPR-Cas12a knockdown in zebrafish embryos led to gross developmental and body axis abnormalities, disrupted ciliogenesis, and upregulated WNT signaling, complementing our human cell studies. This work highlights a conserved and essential biological role of the minor spliceosome in general, and the ZCRB1 protein specifically in cellular and developmental processes across species, shedding light on the multifaceted relationship between splicing regulation, ciliogenesis, and WNT signaling.

Differential alternative splicing analysis links variation in ZRSR2 to a novel type of oral-facial-digital syndrome

PurposeOral-facial-digital (OFD) syndromes are genetically heterogeneous developmental disorders, caused by pathogenic variants in genes involved in primary cilia formation and function. We identified a previously undescribed type of OFD with brain anomalies, ranging from alobar holoprosencephaly to pituitary anomalies, in 6 unrelated families. MethodsExome sequencing of affected probands was supplemented with alternative splicing analysis in patient and control lymphoblastoid and fibroblast cell lines, and primary cilia structure analysis in patient fibroblasts. ResultsIn 1 family with 2 affected males, we identified a germline variant in the last exon of ZRSR2, NM_005089.4:c.1211_1212del NP_005080.1:p.(Gly404GlufsTer23), whereas 7 affected males from 5 unrelated families were hemizygous for the ZRSR2 variant NM_005089.4:c.1207_1208del NP_005080.1:p.(Arg403GlyfsTer24), either occurring de novo or inherited in an X-linked recessive pattern. ZRSR2, located on chromosome Xp22.2, encodes a splicing factor of the minor spliceosome complex, which recognizes minor introns, representing 0.35% of human introns. Patient samples showed significant enrichment of minor intron retention. Among differentially spliced targets are ciliopathy-related genes, such as TMEM107 and CIBAR1. Primary fibroblasts containing the NM_005089.4:c.1207_1208del ZRSR2 variant had abnormally elongated cilia, confirming an association between defective U12-type intron splicing, OFD and abnormal primary cilia formation. ConclusionWe introduce a novel type of OFD associated with elongated cilia and differential splicing of minor intron-containing genes due to germline variation in ZRSR2.

SAP30BP interacts with RBM17/SPF45 to promote splicing in a subset of human short introns

Human pre-mRNA splicing requires the removal of introns with highly variable lengths, from tens to over a million nucleotides. Therefore, mechanisms of intron recognition and splicing are likely not universal. Recently, we reported that splicing in a subset of human short introns with truncated polypyrimidine tracts depends on RBM17 (SPF45), instead of the canonical splicing factor U2 auxiliary factor (U2AF) heterodimer. Here, we demonstrate that SAP30BP, a factor previously implicated in transcriptional control, is an essential splicing cofactor for RBM17. Invitro binding and nuclear magnetic resonance analyses demonstrate that a U2AF-homology motif (UHM) in RBM17 binds directly to a newly identified UHM-ligand motif in SAP30BP. We show that this RBM17-SAP30BP interaction is required to specifically recruit RBM17 to phosphorylated SF3B1 (SF3b155), a U2 small nuclear ribonucleoprotein (U2 snRNP) component in active spliceosomes. We propose a mechanism for splicing in a subset of short introns, in which SAP30BP guides RBM17 in the assembly of active spliceosomes.

Genome-wide detection of human intronic AG-gain variants located between splicing branchpoints and canonical splice acceptor sites.

Human genetic variants that introduce an AG into the intronic region between the branchpoint (BP) and the canonical splice acceptor site (ACC) of protein-coding genes can disrupt pre-mRNA splicing. Using our genome-wide BP database, we delineated the BP-ACC segments of all human introns and found extreme depletion of AG/YAG in the [BP+8, ACC-4] high-risk region. We developed AGAIN as a genome-wide computational approach to systematically and precisely pinpoint intronic AG-gain variants within the BP-ACC regions. AGAIN identified 350 AG-gain variants from the Human Gene Mutation Database, all of which alter splicing and cause disease. Among them, 74% created new acceptor sites, whereas 31% resulted in complete exon skipping. AGAIN also predicts the protein-level products resulting from these two consequences. We performed AGAIN on our exome/genomes database of patients with severe infectious diseases but without known genetic etiology and identified a private homozygous intronic AG-gain variant in the antimycobacterial gene SPPL2A in a patient with mycobacterial disease. AGAIN also predicts a retention of six intronic nucleotides that encode an in-frame stop codon, turning AG-gain into stop-gain. This allele was then confirmed experimentally to lead to loss of function by disrupting splicing. We further showed that AG-gain variants inside the high-risk region led to misspliced products, while those outside the region did not, by two case studies in genes STAT1 and IRF7. We finally evaluated AGAIN on our 14 paired exome-RNAseq samples and found that 82% of AG-gain variants in high-risk regions showed evidence of missplicing. AGAIN is publicly available from https://hgidsoft.rockefeller.edu/AGAIN and https://github.com/casanova-lab/AGAIN.

Effect of human β-Globin second intron on transient gene expression in mammalian cell lines

Exogenous protein expression in mammalian cells is necessary to produce therapeutic proteins and modern medical applications like developing DNA vaccines and gene therapy. This study examines the human-Globin (hBG) second intron's capacity for intron-mediated enhancement (IME) in various mammalian cell lines. Our study's main aim is to investigate the effect of the incorporation and arrangement of the second intron of the human Beta-globin gene into the pVAX-1 expression cassette on improving the expression of foreign genes. Two plasmids were constructed, one with the hBG second intron positioned upstream and the other downstream in the expression cassette. EGFP expression was evaluated at the mRNA and protein levels after transfection using Lipofectamine 2000 using One-way ANOVA analysis. Results showed that the pVAX-1 harbouring the hBG second intron did not lead to enhanced transient EGFP expression and did not exhibit Intron Mediated Enhancement (IME) in tested mammalian cell lines. Further investigations are necessary to understand factors contributing to the lack of enhancement and explore alternative intron options for optimizing foreign gene expression in cell lines.

Combination of MAR and intron increase transgene expression of episomal vectors in CHO cells.

Previous work has shown that the EF-1α promoter of episomal vectors maintains high-level transgene expression in stably transfected Chinese-hamster ovary (CHO) cells. However, the transgene expression levels need to be further increased. Here, we first incorporated matrix attachment regions (MAR), ubiquitous chromatin opening element (UCOE), stabilizing anti repressor elements 40 (STAR 40) elements into episomal vector at different sites and orientations, and systemically assessed their effects on transgene expression in transfected CHO-K1 cells. Results showed that enhanced green fluorescent protein (eGFP) expression levels increased remarkably when MAR X-29 was inserted upstream of the promoter, followed by the insertion of MAR-1 downstream of the poly A, and the orientation had no significant effect. Moreover, MAR X-29 combined with human cytomegalovirus intron (hCMVI) yielded the highest transgene expression levels (4.52-fold). Transgene expression levels were not exclusively dependent on transgene copy numbers and were not related to the mRNA expression level. In addition, vector with MAR X-29+ hCMVI can induce herpes simplex virus thymidine kinase (HSV-TK) protein expression, and the HSV-TK protein showed a cell-killing effect and an obvious bystander effect on HCT116 cells. In conclusion, the combination of MAR X-29 and hCMV intron can achieve high efficiency transgene expression mediated by episomal vectors in CHO-K1 cells. This article is protected by copyright. All rights reserved.

FUBP1 is a general splicing factor facilitating 3′ splice site recognition and splicing of long introns

Splicing of pre-mRNAs critically contributes to gene regulation and proteome expansion in eukaryotes, but our understanding of the recognition and pairing of splice sites during spliceosome assembly lacks detail. Here, we identify the multidomain RNA-binding protein FUBP1 as a key splicing factor that binds to a hitherto unknown cis-regulatory motif. By collecting NMR, structural, and invivo interaction data, we demonstrate that FUBP1 stabilizes U2AF2 and SF1, key components at the 3' splice site, through multivalent binding interfaces located within its disordered regions. Transcriptional profiling and kinetic modeling reveal that FUBP1 is required for efficient splicing of long introns, which is impaired in cancer patients harboring FUBP1 mutations. Notably, FUBP1 interacts with numerous U1 snRNP-associated proteins, suggesting a unique role for FUBP1 in splice site bridging for long introns. We propose a compelling model for 3' splice site recognition of long introns, which represent 80% of all human introns.

Recursive splicing discovery using lariats in total RNA sequencing.

Recursive splicing is a non-canonical splicing mechanism in which an intron is removed in segments via multiple splicing reactions. Relatively few recursive splice sites have been identified with high confidence in human introns, and more comprehensive analyses are needed to better characterize where recursive splicing happens and whether or not it has a regulatory function. In this study, we use an unbiased approach using intron lariats to search for recursive splice sites in constitutive introns and alternative exons in the human transcriptome. We find evidence for recursive splicing in a broader range of intron sizes than previously reported and detail a new location for recursive splicing at the distal ends of cassette exons. In addition, we identify evidence for the conservation of these recursive splice sites among higher vertebrates and the use of these sites to influence alternative exon exclusion. Together, our data demonstrate the prevalence of recursive splicing and its potential influence on gene expression through alternatively spliced isoforms.

Diverse logics and grammar encode notochord enhancers.

The notochord is a defining feature of all chordates. The transcription factors Zic and ETS regulate enhancer activity within the notochord. We conduct high-throughput screens of genomic elements within developing Ciona embryos to understand how Zic and ETS sites encode notochord activity. Our screen discovers an enhancer located near Lama, a gene critical for notochord development. Reversing the orientation of an ETS site within this enhancer abolishes expression, indicating that enhancer grammar is critical for notochord activity. Similarly organized clusters of Zic and ETS sites occur within mouse and human Lama1 introns. Within a Brachyury (Bra) enhancer, FoxA and Bra, in combination with Zic and ETS binding sites, are necessary and sufficient for notochord expression. This binding site logic also occurs within other Ciona and vertebrate Bra enhancers. Collectively, this study uncovers the importance of grammar within notochord enhancers and discovers signatures of enhancer logic and grammar conserved across chordates.

The evolutionary dynamics that retain long neutral genomic sequences in face of indel deletion bias: a model and its application to human introns.

Insertions and deletions (indels) of short DNA segments are common evolutionary events. Numerous studies showed that deletions occur more often than insertions in both prokaryotes and eukaryotes. It raises the question why neutral sequences are not eradicated from the genome. We suggest that this is due to a phenomenon we term border-induced selection. Accordingly, a neutral sequence is bordered between conserved regions. Deletions occurring near the borders occasionally protrude to the conserved region and are thereby subject to strong purifying selection. Thus, for short neutral sequences, an insertion bias is expected. Here, we develop a set of increasingly complex models of indel dynamics that incorporate border-induced selection. Furthermore, we show that short conserved sequences within the neutrally evolving sequence help explain: (i) the presence of very long sequences; (ii) the high variance of sequence lengths; and (iii) the possible emergence of multimodality in sequence length distributions. Finally, we fitted our models to the human intron length distribution, as introns are thought to be mostly neutral and bordered by conserved exons. We show that when accounting for the occurrence of short conserved sequences within introns, we reproduce the main features, including the presence of long introns and the multimodality of intron distribution.

Profiling lariat intermediates reveals genetic determinants of early and late co-transcriptional splicing

Long introns with short exons in vertebrate genes are thought to require spliceosome assembly across exons (exon definition), rather than introns, thereby requiring transcription of an exon to splice an upstream intron. Here, we developed CoLa-seq (co-transcriptional lariat sequencing) to investigate the timing and determinants of co-transcriptional splicing genome wide. Unexpectedly, 90% of all introns, including long introns, can splice before transcription of a downstream exon, indicating that exon definition is not obligatory for most human introns. Still, splicing timing varies dramatically across introns, and various genetic elements determine this variation. Strong U2AF2 binding to the polypyrimidine tract predicts early splicing, explaining exon definition-independent splicing. Together, our findings question the essentiality of exon definition and reveal features beyond intron and exon length that are determinative for splicing timing.

Multi-omics characterization of productive HPV integration in cervical cancer.

e17508 Background: Cervical cancer (CC), which is caused by high-risk human papillomavirus (HPV), remains a significant public health problem worldwide. HPV integration into the host genome is a critical aetiological event in cervical carcinogenesis and progression and it can be silent or actively transcribed that leads to production of viral-host fusion transcripts. We aim to study the characterization of silent and productive HPV integration in cervical cancer. Methods: Cervical cancer (CC), which is caused by high-risk human papillomavirus (HPV), remains a significant public health problem worldwide. HPV integration into the host genome is a critical aetiological event in cervical carcinogenesis and progression and it can be silent or actively transcribed that leads to production of viral-host fusion transcripts. We aim to study the characterization of silent and productive HPV integration in cervical cancer. Results: Virus capture sequencing revealed 762 virus-host DNA fusion breakpoints and only 142 (18.6%) were actively transcribed into fusion transcripts. The productive HPV integrations were nonrandom, with an increase in breakpoints in the HPV E1 region、 human introns and common fragile sites. They were associated with significant changes in host focal genome structure and gene transcription, but not in protein production in 40 kb region, and produced higher levels of E6 and E7 proteins detected by Western Blot. Clinical, transcriptional, proteomic, phosphoproteomic and single-cell data demonstrated that EMT, proliferation and extracellular matrix associated pathway were enriched in productive HPV integrated tumors and contributed to advanced clinical stage. Conclusions: We demonstrated that productive HPV integration is associated with higher E6/E7 proteins and enhanced tumor aggressiveness and immunoevasion in situ to advanced disease. This study improves our understanding of the effects of HPV fusion transcripts on the biology and pathophysiology of HPV-driven cervical cancer that has the potential to direct new prevention and therapeutic strategies

A Quality Control Mechanism of Splice Site Selection Abrogated under Stress and in Cancer

Simple SummarySplicing and alternative splicing play a major role in regulating gene expression, and mis-regulation of splicing can lead to several diseases, including cancer. The aim of this review is to summarize the current knowledge of a quality control mechanism of splice site selection termed Suppression of Splicing (SOS), proposed to protect cells from splicing at the numerous intronic unused 5′ splice sites, and emphasize its relevance to cancer. This relevance stems from the finding that SOS is abrogated under stress and in cancer resulting in the expression of thousands of aberrant nonsense mRNAs that may be toxic to cells. These findings highlight the unexplored potential of such aberrant isoforms as novel targets for cancer diagnosis and therapies.Latent 5’ splice sites, highly abundant in human introns, are not normally used. This led to the proposal of a quality control mechanism, Suppression of Splicing (SOS), which protects cells from splicing at the numerous intronic latent sites, and whose activation can generate nonsense mRNAs. SOS was shown to be independent of Nonsense-Mediated mRNA Decay (NMD). Efforts to decipher the SOS mechanism revealed a pivotal role for initiator-tRNA, independent of protein translation. Recently, nucleolin (a multifunctional protein) was found to directly and specifically bind the initiator-tRNA in the nucleus and was shown to be a protein component of SOS, enabling an updated model of the SOS mechanism. Importantly, SOS is abrogated under stress and in cancer (e.g., in breast cancer cells and gliomas), generating thousands of nonsense mRNAs due to activation of latent splicing. The resulting affected human genes cover a variety of functional groups, including genes involved in cell proliferation and differentiation. Furthermore, in oligodendroglioma, the extent of activation of latent splicing increases with the severity of the cancer. Interesting examples are genes expressing aberrant nonsense mRNAs in both breast cancer and glioma, due to latent splicing activation. These findings highlight the unexplored potential of such aberrant isoforms as novel targets for cancer diagnosis and therapies.

A novel role for nucleolin in splice site selection

ABSTRACT Latent 5ʹ splice sites, not normally used, are highly abundant in human introns, but are activated under stress and in cancer, generating thousands of nonsense mRNAs. A previously proposed mechanism to suppress latent splicing was shown to be independent of NMD, with a pivotal role for initiator-tRNA independent of protein translation. To further elucidate this mechanism, we searched for nuclear proteins directly bound to initiator-tRNA. Starting with UV-crosslinking, we identified nucleolin (NCL) interacting directly and specifically with initiator-tRNA in the nucleus, but not in the cytoplasm. Next, we show the association of initiator-tRNA and NCL with pre-mRNA. We further show that recovery of suppression of latent splicing by initiator-tRNA complementation is NCL dependent. Finally, upon nucleolin knockdown we show activation of latent splicing in hundreds of coding transcripts having important cellular functions. We thus propose nucleolin, a component of the endogenous spliceosome, through its direct binding to initiator-tRNA and its effect on latent splicing, as the first protein of a nuclear quality control mechanism regulating splice site selection to protect cells from latent splicing that can generate defective mRNAs.

TTR exon-humanized mouse optimal for verifying new therapies for FAP

Familial amyloidotic polyneuropathy (FAP) is caused by a mutation in the transthyretin (TTR) gene. In addition, deposition of wild-type TTR can cause senile systemic amyloidosis (SSA). To date, we have produced several transgenic mouse models for FAP and SSA by introducing TTR genes with different promoters or mutations. However, mouse TTR can associate with human TTR to produce hybrid tetramers in transgenic mice. Thus, these transgenic mice cannot be used to test the efficacy of a new therapy. In this study, we attempted to construct an optimized mouse model to verify a new therapy. The TTR gene consists of 4 exons and 3 introns. We prepared two gRNAs, one for the exon 1 and the other for exon 4, and a single donor vector carrying the whole TTR gene in which mouse exons were replaced with human exons. Using these vectors, we produced a TTR exon-humanized mouse with human exons and mouse introns using genome editing technology. These TTR exon-humanized mice showed normal TTR expression patterns in terms of serum TTR level and spatial specificity. These TTR exon-humanized mice will be useful for devising new treatment methods for FAP, including gene therapy.

Discovery of a Subset of Human Short Introns Spliced Out by a Distinct Mechanism

Discovery of a Subset of Human Short Introns Spliced Out by a Distinct Mechanism

Congenital insensitivity to pain: a novel mutation affecting a U12-type intron causes multiple aberrant splicing of SCN9A.

Mutations in the alpha subunit of voltage-gated sodium channel 1.7 (NaV1.7), encoded by SCN9A gene, play an important role in the regulation of nociception and can lead to a wide range of clinical outcomes, ranging from extreme pain syndromes to congenital inability to experience pain. To expand the phenotypic and genotypic spectrum of SCN9A-related channelopathies, we describe the proband, a daughter born from consanguineous parents, who had pain insensitivity, diminished temperature sensation, foot burns, and severe loss of nociceptive nerve fibers in the epidermis. Next-generation sequencing of SCN9A (NM_002977.3) revealed a novel homozygous substitution (c.377+7T>G) in the donor splice site of intron 3. As the RNA functional testing is challenging, the in silico analysis is the first approach to predict possible alterations. In this case, the computational analysis was unable to identify the splicing consensus and could not provide any prediction for splicing defects. The affected intron indeed belongs to the U12 type, a family of introns characterised by noncanonical consensus at the splice sites, accounting only for 0.35% of all human introns, and is not included in most of the training sets for splicing prediction. A functional study on proband RNA showed different aberrant transcripts, where exon 3 was missing and an intron fragment was included. A quantification study using real-time polymerase chain reaction showed a significant reduction of the NaV1.7 canonical transcript. Collectively, these data widen the spectrum of SCN9A-related insensitivity to pain by describing a mutation causing NaV1.7 deficiency, underlying the nociceptor dysfunction, and highlight the importance of molecular investigation of U12 introns' mutations despite the silent prediction.

High frequency of intron retention and clustered H3K4me3-marked nucleosomes in short first introns of human long non-coding RNAs

BackgroundIt is established that protein-coding exons are preferentially localized in nucleosomes. To examine whether the same is true for non-coding exons, we analysed nucleosome occupancy in and adjacent to internal exons in genes encoding long non-coding RNAs (lncRNAs) in human CD4+ T cells and K562 cells.ResultsWe confirmed that internal exons in lncRNAs are preferentially associated with nucleosomes, but also observed an elevated signal from H3K4me3-marked nucleosomes in the sequences upstream of these exons. Examination of 200 genomic lncRNA loci chosen at random across all chromosomes showed that high-density regions of H3K4me3-marked nucleosomes, which we term ‘slabs’, are associated with genomic regions exhibiting intron retention. These retained introns occur in over 50% of lncRNAs examined and are mostly first introns with an average length of just 354 bp, compared to the average length of all human introns of 6355 and 7987 bp in mRNAs and lncRNAs, respectively. Removal of short introns from the dataset abrogated the high upstream H3K4me3 signal, confirming that the association of slabs and short lncRNA introns with intron retention holds genome-wide. The high upstream H3K4me3 signal is also associated with alternatively spliced exons, known to be prominent in lncRNAs. This phenomenon was not observed with mRNAs.ConclusionsThere is widespread intron retention and clustered H3K4me3-marked nucleosomes in short first introns of human long non-coding RNAs, which raises intriguing questions about the relationship of IR to lncRNA function and chromatin organization.

SPF45/RBM17-dependent, but not U2AF-dependent, splicing in a distinct subset of human short introns

Human pre-mRNA introns vary in size from under fifty to over a million nucleotides. We searched for essential factors involved in the splicing of human short introns by screening siRNAs against 154 human nuclear proteins. The splicing activity was assayed with a model HNRNPH1 pre-mRNA containing short 56-nucleotide intron. We identify a known alternative splicing regulator SPF45 (RBM17) as a constitutive splicing factor that is required to splice out this 56-nt intron. Whole-transcriptome sequencing of SPF45-deficient cells reveals that SPF45 is essential in the efficient splicing of many short introns. To initiate the spliceosome assembly on a short intron with the truncated poly-pyrimidine tract, the U2AF-homology motif (UHM) of SPF45 competes out that of U2AF65 (U2AF2) for binding to the UHM-ligand motif (ULM) of the U2 snRNP protein SF3b155 (SF3B1). We propose that splicing in a distinct subset of human short introns depends on SPF45 but not U2AF heterodimer.

Abstract 256: Sequence conservation of BRCA1 intron-exon junctions

Abstract Introduction: Clinical sequencing of BRCA1 and BRCA2 exons and intron-exon boundaries reveals sequence variants of uncertain clinical significance (VUSs). Many BRCA1/2 sequence variants near intron-exon boundaries are potentially pathogenic by disrupting normal splicing patterns. However, molecular analysis of these potential effects is complicated by naturally occurring alternative splicing products. It is not yet clear which of these are required for normal gene functions. We therefore aligned intron-exon boundary sequence strings for 21 BRCA1 exons in order to 1) determine whether VUSs near intron-exon boundaries disrupt conserved sequences, and 2) determine whether sequences flanking exons involved in alternative splicing events are sufficiently conserved. Methods: Sequence strings representing intron-exon junction regions were prepared for 21 BRCA1 exons. Each string consisted of 20 bases of 5' intron, the 5'-most 20 bases of each exon, the 3'-most 20 bases of each exon, and 20 bases of the 3' intron. Strings were similarly prepared for chimpanzee (Pan troglodytes), bonobo (Pan paniscus), gorilla (Gorilla gorilla), orangutan (Pongo abelii), baboon (Papio anubis), and Rhesus macaque (Macaca mulatta). Each string was aligned using the MUSCLE multiple sequence alignment method. Results: Splice junction sequence conservation was from 68%-100% conserved (mean % conservation, 92.7%; median, 95%). In almost all cases, sequence differences were transitions or differing numbers of thymines in poly-T tracts. The two most divergent intron-exon junction regions flanked exons 17 and 18 (70% and 68% similar, respectively). The human exon 16-17 junction is not conserved in in gorilla, and the human exon 18-19 junction is not conserved in macaques. These differences account for the divergence among primates at these sites. Seven alternative splicing events in human BRCA1 are classified as “predominant,” and all are predicted to retain the translational reading frame. All of these alternative splicing events occur in nearly perfectly conserved splicing environments, suggesting they may occur in other species as well and possibly contribute to gene function. Seven clinically identified VUSs that fall within the human BRCA1 splice junction region are wild type base differences in other primates. c.231G>A from human exon 5 is seen in baboon, c.441+18C>T in human intron 6 is seen in orangutan, c.455T>C in exon 7 is seen in orangutan, c.547+7G>A in intron 7 is seen in macaque and baboon, and c.4097-10G>A, c.4097-20C>T, and c.4115G>A, all in or near exon 11, are seen in macaque, baboon, and orangutan. These observations suggest these VUSs are likely to be benign polymorphisms and not pathogenic variants. We note, however, that c.547+7G>A disrupts a candidate intron splicing enhancer as predicted by RegRNA 2.0 and believe this variant warrants further study. Citation Format: Talia Ishfaq, Maryam Salik, Uzma Jafry, Zaain Ahmad, James D. Fackenthal. Sequence conservation of BRCA1 intron-exon junctions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 256.