Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. About half of the 1002 Arabidopsis and 923 human exitrons have sizes of multiples of 3 nucleotides (nt). Splicing of these exitrons results in internally deleted proteins and affects protein domains, disordered regions, and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Intriguingly, annotated intronless genes can be also alternatively spliced via exitron usage. We demonstrate that at least some exitrons originate from ancestral coding exons. Based on our findings, we propose a "splicing memory" hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Altogether, our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals, complementing the repertoire of AS events.

The interplay between epigenetic modification and chromatin compaction is implicated in the regulation of gene expression, and it comprises one of the most fascinating frontiers in cell biology. Although a complete picture is still lacking, it is generally accepted that the differentiation of embryonic stem (ES) cells is accompanied by a selective condensation into heterochromatin with concomitant gene silencing, leaving access only to lineage-specific genes in the euchromatin. ES cells have been reported to have less condensed chromatin, as they are capable of differentiating into any cell type. However, pluripotency itself-even prior to differentiation-is a split state comprising a naïve state and a state in which ES cells prime for differentiation. Here, we show that naïve ES cells decondense their chromatin in the course of downregulating the pluripotency marker Nanog before they initiate lineage commitment. We used fluorescence recovery after photobleaching, and histone modification analysis paired with a novel, to our knowledge, optical stretching method, to show that ES cells in the naïve state have a significantly stiffer nucleus that is coupled to a globally more condensed chromatin state. We link this biophysical phenotype to coinciding epigenetic differences, including histone methylation, and show a strong correlation of chromatin condensation and nuclear stiffness with the expression of Nanog. Besides having implications for transcriptional regulation and embryonic cell sorting and suggesting a putative mechanosensing mechanism, the physical differences point to a system-level regulatory role of chromatin in maintaining pluripotency in embryonic development.

The nucleosomal landscape of chromatin depends on the concerted action of chromatin remodelers. The INO80 remodeler specifically places nucleosomes at the boundary of gene regulatory elements, which is proposed to be the result of an ATP-dependent nucleosome sliding activity that is regulated by extranucleosomal DNA features. Here, we use cryo–electron microscopy and functional assays to reveal how INO80 binds and is regulated by extranucleosomal DNA. Structures of the regulatory A-module bound to DNA clarify the mechanism of linker DNA binding. The A-module is connected to the motor unit via an HSA/post-HSA lever element to chemomechanically couple the motor and linker DNA sensing. Two notable sites of curved DNA recognition by coordinated action of the four actin/actin-related proteins and the motor suggest how sliding by INO80 can be regulated by extranucleosomal DNA features. Last, the structures clarify the recruitment of YY1/Ies4 subunits and reveal deep architectural similarities between the regulatory modules of INO80 and SWI/SNF complexes.

Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits impacts polymerization dynamics and microtubule function. When cells sense excess free tubulin, they trigger degradation of the encoding mRNAs, which requires recognition of the nascent polypeptide by the tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates decay of tubulin mRNAs is unknown. Here, our biochemical and structural analysis reveals that TTC5 recruits the poorly studied protein SCAPER to the ribosome. SCAPER in turn engages the CCR4-NOT deadenylase complex through its CNOT11 subunit to trigger tubulin mRNA decay. SCAPER mutants that cause intellectual disability and retinitis pigmentosa in humans are impaired in CCR4-NOT recruitment, tubulin mRNA degradation, and microtubule-dependent chromosome segregation. Our findings demonstrate how recognition of a nascent polypeptide on the ribosome is physically linked to mRNA decay factors via a relay of protein-protein interactions, providing a paradigm for specificity in cytoplasmic gene regulation.