The tumor suppressor p53 and its antagonists MDM2 and MDM4 integrate stress signaling. For instance, dysbalanced assembly of ribosomes in nucleoli induces p53. Here, we show that the ribosomal protein L22 (RPL22; eL22), under conditions of ribosomal and nucleolar stress, promotes the skipping of MDM4 exon 6. Upon L22 depletion, more full-length MDM4 is maintained, leading to diminished p53 activity and enhanced cellular proliferation. L22 binds to specific RNA elements within intron 6 of MDM4 that correspond to a stem-loop consensus, leading to exon 6 skipping. Targeted deletion of these intronic elements largely abolishes L22-mediated exon skipping and re-enables cell proliferation, despite nucleolar stress. L22 also governs alternative splicing of the L22L1 (RPL22L1) and UBAP2L mRNAs. Thus, L22 serves as a signaling intermediate that integrates different layers of gene expression. Defects in ribosome synthesis lead to specific alternative splicing, ultimately triggering p53-mediated transcription and arresting cell proliferation.

UNASSIGNED: The U1 small nuclear RNA (snRNA) forms ribonucleoprotein particles (RNPs) such as U1 snRNP and U1-TAF15 snRNP. U1 snRNP is one of the most studied RNPs due to its critical role in pre-mRNA splicing in defining the 5\' splice site (5\'ss) of every exon through direct interactions with sequences at exon/intron junctions. Recent reports support the role of U1 snRNP in all steps of transcription, namely initiation, elongation, and termination. Functions of U1-TAF15 snRNP are less understood, though it associates with the transcription machinery and may modulate pre-mRNA splicing by interacting with the 5\'ss and/or 5\'ss-like sequences within the pre-mRNA. An anti-U1 antisense oligonucleotide (ASO) that sequesters the 5\' end of U1 snRNA inhibits the functions of U1 snRNP, including transcription and splicing. However, it is not known if the inhibition of U1 snRNP influences post-transcriptional regulation of pre-mRNA splicing through deep intronic sequences.
UNASSIGNED: We examined the effect of an anti-U1 ASO that sequesters the 5\' end of U1 snRNA on transcription and splicing of all internal exons of the spinal muscular atrophy (SMA) genes, SMN1 and SMN2. Our study was enabled by the employment of a multi-exon-skipping detection assay (MESDA) that discriminates against prematurely terminated transcripts. We employed an SMN2 super minigene to determine if anti-U1 ASO differently affects splicing in the context of truncated introns.
UNASSIGNED: We observed substantial skipping of multiple internal exons of SMN1 and SMN2 triggered by anti-U1 treatment. Suggesting a role for U1 snRNP in interacting with deep intronic sequences, early exons of the SMN2 super minigene with truncated introns were resistant to anti-U1 induced skipping. Consistently, overexpression of engineered U1 snRNAs targeting the 5\'ss of early SMN1 and SMN2 exons did not prevent exon skipping caused by anti-U1 treatment.
UNASSIGNED: Our results uncover a unique role of the U1 snRNA-associated RNPs in splicing regulation executed through deep intronic sequences. Findings are significant for developing novel therapies for SMA based on deep intronic targets.

Posttranscriptional regulation comprises those mechanisms occurring after the initial copy of the DNA sequence is transcribed into an intermediate RNA molecule (i.e., messenger RNA) until such a molecule is used as a template to generate a protein. A subset of these posttranscriptional regulatory mechanisms essentially are destined to process the immature mRNA toward its mature form, conferring the adequate mRNA stability, providing the means for pertinent introns excision, and controlling mRNA turnover rate and quality control check. An additional layer of complexity is added in certain cases, since discrete nucleotide modifications in the mature RNA molecule are added by RNA editing, a process that provides large mature mRNA diversity. Moreover, a number of posttranscriptional regulatory mechanisms occur in a cell- and tissue-specific manner, such as alternative splicing and noncoding RNA-mediated regulation. In this chapter, we will briefly summarize current state-of-the-art knowledge of general posttranscriptional mechanisms, while major emphases will be devoted to those tissue-specific posttranscriptional modifications that impact on cardiac development and congenital heart disease.

Alternative splicing (AS) is an important mechanism contributing to stress-induced regulation of gene expression and proteome diversity. Massive sequencing technologies allow the identification of transcripts generated via stress-responsive AS, potentially important for adaptation to stress conditions. Several bioinformatics tools have been developed to identify differentially expressed alternative splicing events/transcripts from RNA-sequencing results. This chapter describes a detailed protocol for differential alternative splicing analysis using the rMATS tool. In addition, we provide guidelines for validation of the detected splice variants by qRT-PCR based on the obtained output files.

Post-transcriptional regulation by RNA binding proteins can determine gene expression levels and drive changes in cancer cell proteomes. Identifying mechanisms of protein-RNA binding, including preferred sequence motifs bound in vivo, provides insights into protein-RNA networks and how they impact mRNA structure, function, and stability. In this review, we will focus on proteins that bind to AU-rich elements (AREs) in nascent or mature mRNA where they play roles in response to stresses encountered by cancer cells. ARE-binding proteins (ARE-BPs) specifically impact alternative splicing, stability, decay and translation, and formation of RNA-rich biomolecular condensates like cytoplasmic stress granules (SGs). For example, recent findings highlight the role of ARE-BPs - like TIAR and HUR - in chemotherapy resistance and in translational regulation of mRNAs encoding pro-inflammatory cytokines. We will discuss emerging evidence that different modes of ARE-BP activity impact leukaemia and lymphoma development, progression, adaptation to microenvironment and chemotherapy resistance.

Neurodegeneration with brain iron accumulation (NBIA) is a clinically and genetically heterogeneous disease characterized by increased iron deposition in the basal ganglia and progressive degeneration of the nervous system in adulthood. However, in early childhood, there were no characteristic features to perform early diagnosis. In our study, a female child exhibited global developmental delay, intellectual disability, and febrile seizure without other distinct clinical phenotypes. Through whole exome sequencing (WES), a de novo nonsense mutation (c.726C > G, p. Tyr242Ter) of WDR45 gene was identified in this child. She was finally diagnosed as β-propeller protein-associated neurodegeneration (BPAN), one of the recently identified subtypes of NBIA. This mutation could act as a premature stop codon (PSC) which rendered the mutated transcripts to be degraded by nonsense-mediated mRNA decay (NMD), leading to decreased levels of PSC-containing mRNAs. Additionally, through mini-gene splicing assays, this mutation could result in an unprecedented novel transcript with the exon 9 of WDR45 excluded by nonsense-associated splicing alteration (NASA). Transcriptome sequencing (RNA-seq) on total RNAs from PBMCs of the trio revealed three types of alternative splicing events in the patient. Further research implied that downregulation of iron transport genes (TFRC, TFR2, SCARA5) might be the underlying mechanism for the iron accumulation in patients with deficient WDR45. This is the first report about NASA happening in WDR45. It implies that nonsense mutations approximal to splicing sites could affect the disease pathogenesis through more than one molecular mechanism and should be taken into consideration when conducting genetic counseling.

Pre-mRNA splicing is catalyzed in two steps: 5\' splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (1st and 2nd step factors). We recently identified Fyv6 (FAM192A in humans) as a 2nd step factor in S. cerevisiae; however, we did not determine how widespread Fyv6\'s impact is on the transcriptome. To answer this question, we have used RNA-seq to analyze changes in splicing. These results show that loss of Fyv6 results in activation of non-consensus, branch point (BP) proximal 3\' SS transcriptome-wide. To identify the molecular basis of these observations, we determined a high-resolution cryo-EM structure of a yeast product complex spliceosome containing Fyv6 at 2.3 Å. The structure reveals that Fyv6 is the only 2nd step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the 1st step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for fyv6Δ suppressor mutations. The combined transcriptomic, structural, and genetic studies allow us to propose a model in which Yju2/Fyv6 exchange facilitates exon ligation and Fyv6 promotes usage of consensus, BP distal 3\' SS.

SRSF1 is the founding member of the SR protein family. It is required-interchangeably with other SR proteins-for pre-mRNA splicing in vitro, and it regulates various alternative splicing events. Dysregulation of SRSF1 expression contributes to cancer and other pathologies. Here, we characterized SRSF1\'s interactome using proximity labeling and mass spectrometry. This approach yielded 190 proteins enriched in the SRSF1 samples, independently of the N- or C-terminal location of the biotin-labeling domain. The detected proteins reflect established functions of SRSF1 in pre-mRNA splicing and reveal additional connections to spliceosome proteins, in addition to other recently identified functions. We validated a robust interaction with the spliceosomal RNA helicase DDX23/PRP28 using bimolecular fluorescence complementation and in vitro binding assays. The interaction is mediated by the N-terminal RS-like domain of DDX23 and both RRM1 and the RS domain of SRSF1. During pre-mRNA splicing, DDX23\'s ATPase activity is essential for the pre-B to B spliceosome complex transition and for release of U1 snRNP from the 5\' splice site. We show that the RS-like region of DDX23\'s N-terminal domain is important for spliceosome incorporation, while larger deletions in this domain alter subnuclear localization. We discuss how the identified interaction of DDX23 with SRSF1 and other SR proteins may be involved in the regulation of these processes.

Pre-mRNA splicing plays a key role in the regulation of gene expression. Recent discoveries suggest that defects in pre-mRNA splicing, resulting from the dysfunction of certain splicing factors, can impact the expression of genes crucial for genome surveillance mechanisms, including those involved in cellular response to DNA damage. In this study, we analyzed how cells with a non-functional spliceosome-associated Gpl1-Gih35-Wdr83 complex respond to DNA damage. Additionally, we investigated the role of this complex in regulating the splicing of factors involved in DNA damage repair. Our findings reveal that the deletion of any component within the Gpl1-Gih35-Wdr83 complex leads to a significant accumulation of unspliced pre-mRNAs of DNA repair factors. Consequently, mutant cells lacking this complex exhibit increased sensitivity to DNA-damaging agents. These results highlight the importance of the Gpl1-Gih35-Wdr83 complex in regulating the expression of DNA repair factors, thereby protecting the stability of the genome following DNA damage.

Splice site recognition is essential for defining the transcriptome. Drugs like risdiplam and branaplam change how U1 snRNP recognizes particular 5\' splice sites (5\'SS) and promote U1 snRNP binding and splicing at these locations. Despite the therapeutic potential of 5\'SS modulators, the complexity of their interactions and snRNP substrates have precluded defining a mechanism for 5\'SS modulation. We have determined a sequential binding mechanism for modulation of -1A bulged 5\'SS by branaplam using a combination of ensemble kinetic measurements and colocalization single molecule spectroscopy (CoSMoS). Our mechanism establishes that U1-C protein binds reversibly to U1 snRNP, and branaplam binds to the U1 snRNP/U1-C complex only after it has engaged a -1A bulged 5\'SS. Obligate orders of binding and unbinding explain how reversible branaplam interactions cause formation of long-lived U1 snRNP/5\'SS complexes. Branaplam is a ribonucleoprotein, not RNA duplex alone, targeting drug whose action depends on fundamental properties of 5\'SS recognition.