This study investigates the evolutionary dynamics of gene and isoform regulation in mammalian tissues. Researchers sequenced cDNA from 9 tissues across 5 vertebrates (4 mammals and 1 bird) in biological triplicate, revealing that tissue-specific gene expression programs are largely conserved, but alternative splicing is conserved in only a subset of tissues and is frequently lineage-specific. Thousands of novel, lineage-specific and conserved alternative exons were identified, with widely conserved alternative exons showing signatures of binding by splicing factors such as MBNL, PTB, RBFOX, STAR, and TIA, suggesting they are ancestral mammalian splicing regulators. The study also indicates that alternative splicing often alters protein phosphorylation, affecting kinase signaling. Alternative pre-mRNA processing can generate mRNA isoforms with distinct protein products or affect mRNA stability, localization, or translation. It can also produce nonfunctional mRNAs targeted for degradation. Most human alternative splicing is tissue-regulated, but the extent of conservation across mammalian species has not been fully studied. The study used RNA-Seq to analyze 9 tissues from 5 vertebrates, including rodents, rhesus macaques, cows, and chickens. The data showed that most tissues have conserved expression signatures, with some exceptions indicating species-specific divergence at a phylogenetic distance of ~300 MY. Some tissues have conserved splicing signatures, with brain and heart/muscle showing strong splicing conservation between mammals and chickens. Other tissues, such as colon, kidney, liver, lung, and spleen, exhibited species-dominated clustering, suggesting less conserved splicing patterns. Lineage-specific changes in splicing factor expression may explain the tendency for splicing patterns to cluster by species rather than tissue. Conserved, tissue-specific alternative exons were identified, with examples like EEF1δ showing highly conserved splicing patterns. The study found that alternative splicing events are often conserved across species, with some exons alternatively spliced in all mammals studied. Exons that were constitutively spliced in a lineage-specific manner and alternatively spliced elsewhere were identified, representing losses of alternative splicing. The study also found that exons with nearby G-runs were more likely to have converted from constitutive to alternative splicing. Splice site changes may convert alternative to constitutive splicing, with exons that recently converted from constitutive to alternative splicing having weaker splice sites. Exons that converted from alternative to constitutive splicing showed increased turnover of splicing regulatory elements. Tissue-specific regulatory motifs accumulated in broadly alternative exons, with strong conservation of motifs associated with splicing factors like MBNL, PTB, RBFOX, STAR, and TIA. Alternative splicing alters phosphorylation potential, with conserved, tissue-specific exons showing higher phosphorylation site density. The study found that tissue-specific alternative splicing can delimit the scope of kinase signaling, withThis study investigates the evolutionary dynamics of gene and isoform regulation in mammalian tissues. Researchers sequenced cDNA from 9 tissues across 5 vertebrates (4 mammals and 1 bird) in biological triplicate, revealing that tissue-specific gene expression programs are largely conserved, but alternative splicing is conserved in only a subset of tissues and is frequently lineage-specific. Thousands of novel, lineage-specific and conserved alternative exons were identified, with widely conserved alternative exons showing signatures of binding by splicing factors such as MBNL, PTB, RBFOX, STAR, and TIA, suggesting they are ancestral mammalian splicing regulators. The study also indicates that alternative splicing often alters protein phosphorylation, affecting kinase signaling. Alternative pre-mRNA processing can generate mRNA isoforms with distinct protein products or affect mRNA stability, localization, or translation. It can also produce nonfunctional mRNAs targeted for degradation. Most human alternative splicing is tissue-regulated, but the extent of conservation across mammalian species has not been fully studied. The study used RNA-Seq to analyze 9 tissues from 5 vertebrates, including rodents, rhesus macaques, cows, and chickens. The data showed that most tissues have conserved expression signatures, with some exceptions indicating species-specific divergence at a phylogenetic distance of ~300 MY. Some tissues have conserved splicing signatures, with brain and heart/muscle showing strong splicing conservation between mammals and chickens. Other tissues, such as colon, kidney, liver, lung, and spleen, exhibited species-dominated clustering, suggesting less conserved splicing patterns. Lineage-specific changes in splicing factor expression may explain the tendency for splicing patterns to cluster by species rather than tissue. Conserved, tissue-specific alternative exons were identified, with examples like EEF1δ showing highly conserved splicing patterns. The study found that alternative splicing events are often conserved across species, with some exons alternatively spliced in all mammals studied. Exons that were constitutively spliced in a lineage-specific manner and alternatively spliced elsewhere were identified, representing losses of alternative splicing. The study also found that exons with nearby G-runs were more likely to have converted from constitutive to alternative splicing. Splice site changes may convert alternative to constitutive splicing, with exons that recently converted from constitutive to alternative splicing having weaker splice sites. Exons that converted from alternative to constitutive splicing showed increased turnover of splicing regulatory elements. Tissue-specific regulatory motifs accumulated in broadly alternative exons, with strong conservation of motifs associated with splicing factors like MBNL, PTB, RBFOX, STAR, and TIA. Alternative splicing alters phosphorylation potential, with conserved, tissue-specific exons showing higher phosphorylation site density. The study found that tissue-specific alternative splicing can delimit the scope of kinase signaling, with