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Wild relatives of crops thrive in habitats where environmental conditions can be restrictive for productivity and survival of cultivated species. The genetic basis of this variability, particularly for tolerance to high temperatures, is not well understood. We examined the capacity of wild and cultivated accessions to acclimate to rapid temperature elevations that cause heat stress (HS). We investigated genotypic variation in thermotolerance of seedlings of wild and cultivated accessions. The contribution of polymorphisms associated with thermotolerance variation was examined regarding alterations in function of the identified gene. We show that tomato germplasm underwent a progressive loss of acclimation to strong temperature elevations. Sensitivity is associated with intronic polymorphisms in the HS transcription factor HsfA2 which affect the splicing efficiency of its pre-mRNA. Intron splicing in wild species results in increased synthesis of isoform HsfA2-II, implicated in the early stress response, at the expense of HsfA2-I which is involved in establishing short-term acclimation and thermotolerance. We propose that the selection for modern HsfA2 haplotypes reduced the ability of cultivated tomatoes to rapidly acclimate to temperature elevations, but enhanced their short-term acclimation capacity. Hence, we provide evidence that alternative splicing has a central role in the definition of plant fitness plasticity to stressful conditions.

In mammalians and yeast, the splicing factor U2AF65/Mud2p functions in precursor messenger RNA (pre-mRNA) processing. Arabidopsis AtU2AF65b encodes a putative U2AF65 but its specific functions in plants are unknown. This paper examines the function of AtU2AF65b as a negative regulator of flowering time in Arabidopsis. We investigated the expression and function of AtU2AF65b in abscisic acid (ABA)-regulated flowering as well as the transcript abundance and pre-mRNA splicing of flowering-related genes in the knock-out mutants of AtU2AF65b. The atu2af65b mutants show early-flowering phenotype under both long-day and short-day conditions. The transcript accumulation of the flowering repressor gene FLOWERING LOCUS C (FLC) is reduced in the shoot apex of atu2af65b, due to both increased intron retention and reduced transcription activation. Reduced transcription of FLC results, at least partially, from the abnormal splicing and reduced transcript abundance of ABSCISIC ACID-INSENSITIVE 5 (ABI5), which encodes an activator of FLC in ABA-regulated flowering signaling. Additionally, the expression of AtU2AF65b is promoted by ABA. Transition to flowering and splicing of FLC and ABI5 in the atu2af65b mutants are compromised during ABA-induced flowering. ABA-responsive AtU2AF65b functions in the pre-mRNA splicing of FLC and ABI5 in shoot apex, whereby AtU2AF65b is involved in ABA-mediated flowering transition in Arabidopsis.

Summary Heat stress significantly impacts global rice production, highlighting the critical need to understand the genetic basis of heat resistance in rice. U2AF (U2 snRNP auxiliary factor) is an essential splicing complex with critical roles in recognizing the 3′‐splice site of precursor messenger RNAs (pre‐mRNAs). The U2AF small subunit (U2AF35) can bind to the 3′‐AG intron border and promote U2 snRNP binding to the branch‐point sequences of introns through interaction with the U2AF large subunit (U2AF65). However, the functions of U2AF35 in plants are poorly understood. In this study, we discovered that the OsU2AF35a gene was vigorously induced by heat stress and could positively regulate rice thermotolerance during both the seedling and reproductive growth stages. OsU2AF35a interacts with OsU2AF65a within the nucleus, and both of them can form condensates through liquid–liquid phase separation (LLPS) following heat stress. The intrinsically disordered regions (IDR) are accountable for their LLPS. OsU2AF35a condensation is indispensable for thermotolerance. RNA‐seq analysis disclosed that, subsequent to heat treatment, the expression levels of several genes associated with water deficiency and oxidative stress in osu2af35a‐1 were markedly lower than those in ZH11. In accordance with this, OsU2AF35a is capable of positively regulating the oxidative stress resistance of rice. The pre‐mRNAs of a considerable number of genes in the osu2af35a‐1 mutant exhibited defective splicing, among which was the OsHSA32 gene. Knocking out OsHSA32 significantly reduced the thermotolerance of rice, while overexpressing OsHSA32 could partially rescue the heat sensitivity of osu2af35a‐1. Together, our findings uncovered the essential role of OsU2AF35a in rice heat stress response through protein separation and regulating alternative pre‐mRNA splicing.

• Protein arginine methyltransferases (PRMTs) regulate many physiological processes, including autophagy. However, the direct roles of the various PRMTs during autophagosome formation remain unclear. Here, we characterised the function of MoHMT1 in the rice blast fungus, Magnaporthe oryzae. • Knockout of MoHMT1 results in inhibited growth and a decreased ability to cause disease lesions on rice seedlings. MoHMT1 catalyses the di-methylation of arginine 247, 251, 261 and 271 residues of MoSNP1, a U1 small nuclear ribonucleoprotein (snRNP) component, likely in a manner dependent on direct interaction. RNA-seq analysis revealed that alternative splicing of pre-mRNAs of 558 genes, including the autophagy-related (ATG) gene MoATG4, was altered in MoHMT1 deletion mutants, compared with wild-type strains under normal growth conditions. • During light exposure or nitrogen starvation, MoHMT1 localises to autophagosomes and MoHMT1 mutants display defects in autophagy induction. Under nitrogen starvation, six additional MoATG genes were identified with retained introns in their mRNA transcripts, corresponding with a significant reduction in transcripts of intron-spliced isoforms in the MoHMT1 mutant strain. • Our study shows that arginine methylation plays an essential role in accurate pre-mRNA splicing necessary for a range of developmental processes, including autophagosome formation.

Biomolecular condensates segregate nuclei into discrete regions, facilitating the execution of distinct biological functions. Here, it is identified that the WW domain containing adaptor with coiled‐coil (WAC) is localized to nuclear speckles via its WW domain and plays a pivotal role in regulating alternative splicing through the formation of biomolecular condensates via its C‐terminal coiled‐coil (CC) domain. WAC acts as a scaffold protein and facilitates the integration of RNA‐binding motif 12 (RBM12) into nuclear speckles, where RBM12 potentially interacts with the spliceosomal U5 small nuclear ribonucleoprotein (snRNP). Importantly, knockdown of RBM12, or deletion of the WAC CC domain led to altered splicing outcomes, resulting in an elevated level of BECN1‐S, the short splice variant of BECN1 that is shown to upregulate mitophagy. Thus, the findings reveal a previously unrecognized mechanism for the nuclear regulation of mitochondrial function through liquid–liquid phase separation (LLPS) and provide insights into the pathogenesis of WAC‐related disorders. WW domain containing adaptor with coiled‐coil (WAC) forms nuclear condensates and regulates alternative splicing through its coiled‐coil domain. WAC scaffolds RBM12, potentially interacting with U5 snRNP. Alterations in WAC affect alternative splicing and elevating BECN1‐S expression which leads to hyperactivated mitophagy.

Recent progress in global sequence and microarray data analysis has revealed the increasing complexity of the human transcriptome. Alternative splicing generates a huge diversity of transcript variants and disruption of splicing regulatory networks is emerging as an important contributor to various diseases, including cancer. Current efforts to establish the dynamic repertoire of transcripts that are generated in health and disease are showing that many cancer‐associated alternative‐splicing events occur in the absence of mutations in the affected genes. A growing body of evidence reveals changes in splicing‐factor expression that correlate with cancer development, progression and response to therapy. Here, we discuss how recent links between cancer and altered expression of proteins implicated in splicing regulation are bringing the splicing machinery to the fore as a potential target for anticancer treatment.

Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45–55 of the DMD gene, might improve patients’ symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45–55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44–56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5′ splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing.

U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5′‐splice site early during spliceosome assembly. It represents a prototype spliceosomal subunit containing a paradigmatic Sm core RNP. The crystal structure of human U1 snRNP obtained from natively purified material by in situ limited proteolysis at 4.4 Å resolution reveals how the seven Sm proteins, each recognize one nucleotide of the Sm site RNA using their Sm1 and Sm2 motifs. Proteins D1 and D2 guide the snRNA into and out of the Sm ring, and proteins F and E mediate a direct interaction between the Sm site termini. Terminal extensions of proteins D1, D2 and B/B′, and extended internal loops in D2 and B/B′ support a four‐way RNA junction and a 3′‐terminal stem‐loop on opposite sides of the Sm core RNP, respectively. On a higher organizational level, the core RNP presents multiple attachment sites for the U1‐specific 70K protein. The intricate, multi‐layered interplay of proteins and RNA rationalizes the hierarchical assembly of U snRNPs in vitro and in vivo . The U1 small nuclear ribonucleoprotein initiates the assembly of the spliceosome. Here, the structure of the natively purified U1 small nuclear ribonucleoprotein particle reveals the core Sm protein ring and its interactions with the Sm site in the small nuclear RNA.

Little is known about the higher‐order structure of prespliceosomal A complexes, in which pairing of the pre‐mRNA's splice sites occurs. Here, human A complexes were isolated under physiological conditions by double‐affinity selection. Purified complexes contained stoichiometric amounts of U1, U2 and pre‐mRNA, and crosslinking studies indicated that these form concomitant base pairing interactions with one another. A complexes contained nearly all U1 and U2 proteins plus ∼50 non‐snRNP proteins. Unexpectedly, proteins of the hPrp19/CDC5 complex were also detected, even when A complexes were formed in the absence of U4/U6 snRNPs, demonstrating that they associate independent of the tri‐snRNP. Double‐affinity purification yielded structurally homogeneous A complexes as evidenced by electron microscopy, and allowed for the first time the generation of a three‐dimensional structure. A complexes possess an asymmetric shape (∼260 × 200 × 195 Å) and contain a main body with various protruding elements, including a head‐like domain and foot‐like protrusions. Complexes isolated here are well suited for in vitro assembly studies to determine factor requirements for the A to B complex transition.

We recently reported that spliceosomes alter messenger ribonucleoprotein particle (mRNP) composition by depositing several proteins 20–24 nucleotides upstream of mRNA exon–exon junctions. When assembled in vitro , this so‐called ‘exon–exon junction complex’ (EJC) contains at least five proteins: SRm160, DEK, RNPS1, Y14 and REF. To better investigate its functional attributes, we now describe a method for generating spliced mRNAs both in vitro and in vivo that either do or do not carry the EJC. Analysis of these mRNAs in Xenopus laevis oocytes revealed that this complex is the species responsible for enhancing nucleocytoplasmic export of spliced mRNAs. It does so by providing a strong binding site for the mRNA export factors REF and TAP/p15. Moreover, by serving as an anchoring point for the factors Upf2 and Upf3, the EJC provides a direct link between splicing and nonsense‐mediated mRNA decay. Finally, we show that the composition of the EJC is dynamic in vivo and is subject to significant evolution upon mRNA export to the cytoplasm.