Lineage-specific splicing regulation of MAPT gene in the primate brain.

Divergence of precursor messenger RNA (pre-mRNA) alternative splicing (AS) is widespread in mammals, including primates, but the underlying mechanisms and functional impact are poorly understood. Here, we modeled cassette exon inclusion in primate brains as a quantitative trait and identified 1,170 (∼3%) exons with lineage-specific splicing shifts under stabilizing selection. Among them, microtubule-associated protein tau (MAPT) exons 2 and 10 underwent anticorrelated, two-step evolutionary shifts in the catarrhine and hominoid lineages, leading to their present inclusion levels in humans. The developmental-stage-specific divergence of exon 10 splicing, whose dysregulation can cause frontotemporal lobar degeneration (FTLD), is mediated by divergent distal intronic MBNL-binding sites. Competitive binding of these sites by CRISPR-dCas13d/gRNAs effectively reduces exon 10 inclusion, potentially providing a therapeutically compatible approach to modulate tau isoform expression. Our data suggest adaptation of MAPT function and, more generally, a role for AS in the evolutionary expansion of the primate brain.

CRISPR-dCas13d-based deep screening of proximal and distal splicing-regulatory elements.

Pre-mRNA splicing, a key process in gene expression, can be therapeutically modulated using various drug modalities, including antisense oligonucleotides (ASOs). However, determining promising targets is hampered by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically inactive CRISPR-RfxCas13d RNA-targeting system (dCas13d/gRNA) as a programmable platform to bind SREs and modulate splicing by competing against endogenous splicing factors. SpliceRUSH, a high-throughput screening method, was developed to map SREs in any gene of interest using a lentivirus gRNA library that tiles the genetic region, including distal intronic sequences. When applied to SMN2, a therapeutic target for spinal muscular atrophy, SpliceRUSH robustly identifies not only known SREs but also a previously unknown distal intronic SRE, which can be targeted to alter exon 7 splicing using either dCas13d/gRNA or ASOs. This technology enables a deeper understanding of splicing regulation with applications for RNA-based drug discovery.

Deep screening of proximal and distal splicing-regulatory elements in a native sequence context.

Pre-mRNA splicing, a key process in gene expression, can be therapeutically modulated using various drug modalities, including antisense oligonucleotides (ASOs). However, determining promising targets is impeded by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically dead CRISPR-RfxCas13d RNA-targeting system (dCas13d/gRNA) as a programmable platform to bind SREs and modulate splicing by competing against endogenous splicing factors. SpliceRUSH, a high-throughput screening method, was developed to map SREs in any gene of interest using a lentivirus gRNA library that tiles the genetic region, including distal intronic sequences. When applied to SMN2, a therapeutic target for spinal muscular atrophy, SpliceRUSH robustly identified not only known SREs, but also a novel distal intronic splicing enhancer, which can be targeted to alter exon 7 splicing using either dCas13d/gRNA or ASOs. This technology enables a deeper understanding of splicing regulation with applications for RNA-based drug discovery.

Auditory sensitivity and vocal acoustics in five species of estrildid songbirds.

Comparative studies of acoustic communication in clades with diverse signal features provide a powerful framework for testing relationships between perception and behaviour. We measured auditory sensitivity in five species of estrildid songbirds with acoustically distinct songs and tested whether differences aligned with species differences in song frequency content. Species were chosen based on phylogeny and differences in song acoustics. Behavioural audiograms were obtained using operant training and testing. Adult audiograms were compared across species and between sexes within a species. Juvenile and adult audiograms were compared in one species. The audiograms of adults reared by their own species and those reared and tutored by another species were compared in one species. Results showed that audiograms were similar across species and similar to previous reports of songbird auditory sensitivity. Species differed in the highest frequency detected and the frequency of peak sensitivity. While hearing frequency range was not correlated with song frequency bandwidth, the frequency of peak sensitivity was highly corelated with the frequency of peak energy in song. Sensitivity did not differ based on sex, age or tutoring experience. Our findings suggest that adaptations in songbird auditory sensitivity are largely constrained by shared peripheral and central encoding mechanisms, with species-specific perception appearing only at peak sensitivity.