Survival of motor neuron 2, centromeric (SMN2) is a gene that modifies the severity of spinal muscular atrophy (SMA), a motor-neuron disease that is the leading genetic cause of infant mortality. Increasing inclusion of SMN2 exon 7, which is predominantly skipped, holds promise to treat or possibly cure SMA; one practical strategy is the disruption of splicing silencers that impair exon 7 recognition. By using an antisense oligonucleotide (ASO)-tiling method, we systematically screened the proximal intronic regions flanking exon 7 and identified two intronic splicing silencers (ISSs): one in intron 6 and a recently described one in intron 7. We analyzed the intron 7 ISS by mutagenesis, coupled with splicing assays, RNA-affinity chromatography, and protein overexpression, and found two tandem hnRNP A1/A2 motifs within the ISS that are responsible for its inhibitory character. Mutations in these two motifs, or ASOs that block them, promote very efficient exon 7 inclusion. We screened 31 ASOs in this region and selected two optimal ones to test in human SMN2 transgenic mice. Both ASOs strongly increased hSMN2 exon 7 inclusion in the liver and kidney of the transgenic animals. Our results show that the high-resolution ASO-tiling approach can identify cis-elements that modulate splicing positively or negatively. Most importantly, our results highlight the therapeutic potential of some of these ASOs in the context of SMA.
hnRNP A1 binds to RNA in a cooperative manner. Initial hnRNP A1 binding to an exonic splicing silencer at the 3 end of human immunodeficiency virus type 1 (HIV-1) tat exon 3, which is a high-affinity site, is followed by cooperative spreading in a 3-to-5 direction. As hnRNP A1 propagates toward the 5 end of the exon, it antagonizes binding of a serine/arginine-rich (SR) protein to an exonic splicing enhancer, thereby inhibiting splicing at that exon's alternative 3 splice site. tat exon 3 and the preceding intron of HIV-1 pre-mRNA can fold into an elaborate RNA secondary structure in solution, which could potentially influence hnRNP A1 binding. We report here that hnRNP A1 binding and splicing repression can occur on an unstructured RNA. Moreover, hnRNP A1 can effectively unwind an RNA hairpin upon binding, displacing a bound protein. We further show that hnRNP A1 can also spread in a 5-to-3 direction, although when initial binding takes place in the middle of an RNA, spreading preferentially proceeds in a 3-to-5 direction. Finally, when two distant high-affinity sites are present on the same RNA, they facilitate cooperative spreading of hnRNP A1 between the two sites.The coding sequences of many eukaryotic genes are interrupted by noncoding introns, which are also present in the primary transcripts, or pre-mRNAs. The introns must be precisely removed, and the coding exons joined, to allow translation of functional proteins. Pre-mRNA splicing, a nuclear process, can be constitutive or alternative. Constitutive splicing is the removal of introns by joining together all the adjacent exons in the order of their arrangement. In constitutive splicing, a single protein is produced from a single pre-mRNA, regardless of where and when the gene is expressed. In alternative splicing, variable use of splice sites allows two or more mature mRNAs to be generated from the same pre-mRNA. For example, an entire exon or part of an exon can be included or skipped in different spliced mRNAs. Alternative splicing is a prevalent way by which many eukaryotes diversify the number of proteins produced from a single pre-mRNA transcript (57,62).Analysis of the human genome indicated that more than 74% of human genes encode at least two isoforms by alternative splicing (27,28,35,48). An extreme example of alternative splicing is the Drosophila Dscam gene, in which a single premRNA transcript apparently encodes 38,016 protein isoforms through combinatorial alternative splicing events (21, 54). Alternative splicing can in many cases be subject to regulation, for example, in a cell-type-specific manner, during embryonic development, or in response to signaling pathways.Retroviruses such as human immunodeficiency virus type 1 (HIV-1) also depend on alternative splicing to produce all of the viral proteins from a single primary transcript (59). The unspliced transcript is necessary for viral replication, packaging into virions, and translation of several proteins, whereas other viral proteins are generated from partially spliced or fully splic...
Background Standard Breast Cancer (BC) risk prediction models based only on epidemiologic factors generally have quite poor performance, and there have been a number of risk scores proposed to improve them, such as AI-based mammographic information, polygenic risk scores and pathogenic variants. Even with these additions BC risk prediction performance is still at best moderate. In that decreased DNA repair capacity (DRC) is a major risk factor for development of cancer, we investigated the potential to improve BC risk prediction models by including a measured phenotypic DRC assay: Methods Using blood samples from the Breast Cancer Family Registry we assessed the performance of phenotypic markers of DRC in 46 matched pairs of individuals, one from each pair with BC (with blood drawn before BC diagnosis) and the other from controls matched by age and time since blood draw. We assessed DRC in thawed cryopreserved peripheral blood mononuclear cells (PBMCs) by measuring γ-H2AX yields (a marker for DNA double-strand breaks) at multiple times from 1 to 20 hrs after a radiation challenge. The studies were performed using surface markers to discriminate between different PBMC subtypes. Results The parameter Fres, the residual damage signal in PBMC B cells at 20 hrs post challenge, was the strongest predictor of breast cancer with an AUC (Area Under receiver-operator Curve) of 0.89 [95% Confidence Interval: 0.84–0.93] and a BC status prediction accuracy of 0.80. To illustrate the combined use of a phenotypic predictor with standard BC predictors, we combined Fres in B cells with age at blood draw, and found that the combination resulted in significantly greater BC predictive power (AUC of 0.97 [95% CI: 0.94–0.99]), an increase of 13 percentage points over age alone. Conclusions If replicated in larger studies, these results suggest that inclusion of a fingerstick-based phenotypic DRC blood test has the potential to markedly improve BC risk prediction.
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