Proteome Diversity Research Articles

N-terminal proteoforms may engage in different protein complexes.

Alternative translation initiation and alternative splicing may give rise to N-terminal proteoforms, proteins that differ at their N-terminus compared with their canonical counterparts. Such proteoforms can have altered localizations, stabilities, and functions. Although proteoforms generated from splice variants can be engaged in different protein complexes, it remained to be studied to what extent this applies to N-terminal proteoforms. To address this, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. First, we generated a catalogue of N-terminal proteoforms found in the HEK293T cellular cytosol from which 22 pairs were selected for interactome profiling. In addition, we provide evidence for the expression of several N-terminal proteoforms, identified in our catalogue, across different human tissues, as well as tissue-specific expression, highlighting their biological relevance. Protein-protein interaction profiling revealed that the overlap of the interactomes for both proteoforms is generally high, showing their functional relation. We also showed that N-terminal proteoforms can be engaged in new interactions and/or lose several interactions compared with their canonical counterparts, thus further expanding the functional diversity of proteomes.

A snapshot of Bothrops jararaca snake venom gland subcellular proteome

Snake venom protein synthesis undergoes finely regulated processes in the specialized secretory epithelium within the venom gland. Such processes occur within a defined period in the cell and at specific cellular locations. Thus, the determination of subcellular proteomes allows the characterization of protein groups for which the site may be relevant to their biological roles, thereby allowing the deconvolution of complex biological circuits into functional information. In this regard, we performed subcellular fractionation of proteins from B. jararaca venom gland, focusing on nuclear proteins since this cellular compartment comprises key effectors that shape gene expression. Our results provided a snapshot of B. jararaca's subcellular venom gland proteome and pointed to a ‘conserved’ proteome core among different life stages (newborn and adult) and between sexes (adult male and female). Overall, the top 15 highly abundant proteins identified in B. jararaca venom glands mirrored the panel of highly expressed genes in human salivary glands. Therefore, the expression profile observed for such a protein set could be considered a conserved core signature of salivary gland secretory epithelium. Moreover, the newborn venom gland displayed a unique expression signature of transcription factors involved in regulating transcription and biosynthetic processes and may mirror biological constraints of the ontogenetic development of B. jararaca, contributing to venom proteome diversity.

Investigating the clinical relevance of RNA editing events and their derived neoantigens in patients with melanoma treated with immunotherapy.

e21580 Background: RNA editing (RE) is a post-transcriptional modification occurring in RNA, which promotes transcriptome and proteome diversity. Peptides translated from RE sites may represent a new source of neoantigens in cancer (RE-derived neoantigens, RED-neoantigens) that could be presented on tumor surface by human-leukocyte-antigen (HLA) and recognized by specific T-cells, leading to an improved response to immune checkpoint inhibitors (ICIs). However, the clinical significance of both RE events and RED-neoantigens in melanoma patients with ICIs therapy has not been systematically characterized. Methods: The WES and transcriptome data with immunotherapy response of melanoma were obtained from the GEO and SRA database. Vardict and VEP were used to identify and annotate the somatic mutations. Polysover was applied to identify the HLA genotype. RNA editing events were identified by Reditools2 and filtered by a custom pipeline. miRTar2GO was implemented to predict the RE event whether located in miRNA targets within the 3' UTR region. NetMHCpan and NetCTLpan were used to identify and characterize RED-neoantigens. K-M survival analysis was performed on the training set to investigate the clinical relevance of RNA editing events and RED-neoantigens in melanoma patients treated with immunotherapy. The independent melanoma dataset was used to validate this clinical relevance. Results: In total, 7,116 RE events were identified, most of which were A-to-I events. 34% of RE sites in 3'UTR and 15% in 5' UTR were located in the seed regions of miRNA targets and transcription factor binding sites (TFBS), respectively, suggesting the different functional effects of the RE events. Furthermore, using our custom pipeline, 631 RED-neoantigens were identified that show a significantly greater peptide-MHC affinity, and facilitate epitope processing and presentation than wild-type peptides. Meanwhile, we found that patients exhibiting a high RED-neoantigens burden correlated with an increased OS, and a significantly higher RED-neoantigens burden was observed in responders. The area under the curve (AUC) of the RED-neoantigen was 0.831 of OS and 0.686 of responsiveness. Conclusions: The findings in this study provide a more insightful view and resource for investigating the neoantigens in cancer as well as for novel cancer immunotherapies and biomarker development.

RNA alternative splicing impacts the risk for alcohol use disorder

Alcohol use disorder (AUD) is a complex genetic disorder characterized by problems arising from excessive alcohol consumption. Identifying functional genetic variations that contribute to risk for AUD is a major goal. Alternative splicing of RNA mediates the flow of genetic information from DNA to gene expression and expands proteome diversity. We asked whether alternative splicing could be a risk factor for AUD. Herein, we used a Mendelian randomization (MR)-based approach to identify skipped exons (the predominant splicing event in brain) that contribute to AUD risk. Genotypes and RNA-seq data from the CommonMind Consortium were used as the training dataset to develop predictive models linking individual genotypes to exon skipping in the prefrontal cortex. We applied these models to data from the Collaborative Studies on Genetics of Alcoholism to examine the association between the imputed cis-regulated splicing outcome and the AUD-related traits. We identified 27 exon skipping events that were predicted to affect AUD risk; six of these were replicated in the Australian Twin-family Study of Alcohol Use Disorder. Their host genes are DRC1, ELOVL7, LINC00665, NSUN4, SRRM2 and TBC1D5. The genes downstream of these splicing events are enriched in neuroimmune pathways. The MR-inferred impacts of the ELOVL7 skipped exon on AUD risk was further supported in four additional large-scale genome-wide association studies. Additionally, this exon contributed to changes of gray matter volumes in multiple brain regions, including the visual cortex known to be involved in AUD. In conclusion, this study provides strong evidence that RNA alternative splicing impacts the susceptibility to AUD and adds new information on AUD-relevant genes and pathways. Our framework is also applicable to other types of splicing events and to other complex genetic disorders.

Intestinal Epithelial Cell-Related Alternative Splicing Events in Dextran Sodium Sulfate-Induced Acute Colitis.

Alternative splicing of pre-messenger RNA is recognized as the crucial mechanism for gene expression regulation and proteome diversity generation. Alternative splicing has been found to be related to the pathogenesis of inflammatory bowel disease. The aim of this study was to identify the alternative splicing events in intestinal epithelial cells from mouse models of acute colitis and expand the understanding of the pathogenesis of inflammatory bowel disease. The acute colitis mouse models were constructed, and intestinal epithelial cells of the colon were isolated for RNA sequence. The replicate Multivariate Analysis of Transcript Splicing software was used to analyze the alternative splicing events. The functional analysis was performed on genes with significant differential alternative splicing events. The alternative splicing events of picked genes were validated by reverse transcription polymerase chain reaction. A total of 340 significant differential alternative splicing events (from 293 genes) were screened out in acute colitis, and the alternative splicing events of CDK5-regulatory subunit associated protein 3 and TRM5 tRNA methyltransferase 5 were validated. The functional analysis showed that differential alternative splicing events in acute colitis participate in the apoptotic process, and the alternative splicing events of 3 genes (BCL2/adenovirus E1B-interacting protein 2, tumor necrosis factor receptor-associated factor 1, and tumor necrosis factor receptor-associated factor 7) were validated by reverse transcription polymerase chain reaction. This study pointed out the potential impact of different alternative splicing in acute colitis.

Mapping splice QTLs reveals distinct transcriptional and post-transcriptional regulatory variation of gene expression and identifies putative alternative splicing variation mediating complex trait variation in pigs

BackgroundAlternative splicing is an important step in gene expression, generating multiple isoforms for the same genes and greatly expanding the diversity of proteomes. Genetic variation in alternative splicing contributes to phenotypic diversity in natural populations. However, the genetic basis of variation in alternative splicing in livestock including pigs remains poorly understood.ResultsIn this study, using a Duroc x Pietrain F2 pig population, we performed genome-wide analysis of alternative splicing estimated from stranded RNA-Seq data in skeletal muscle. We characterized the genetic architecture of alternative splicing and compared its basic features with those of overall gene expression. We detected a large number of novel alternative splicing events that were not previously annotated. We found heritability of quantitative alternative splicing scores (percent spliced in or PSI) to be lower than that of overall gene expression. In addition, heritabilities showed little correlation between alternative splicing and overall gene expression. We mapped expression QTLs (eQTLs) and splice QTLs (sQTLs) and found them to be largely non-overlapping. Finally, we integrated sQTL mapping with phenotype QTL (pQTL mapping to identify potential mediator of pQTL effect by alternative splicing.ConclusionsOur results suggest that regulatory variation exists at multiple levels and that their genetic controls are distinct, offering opportunities for genetic improvement.

Alternative Splicing Changes Promoted by NOVA2 Upregulation in Endothelial Cells and Relevance for Gastric Cancer.

Angiogenesis is crucial for cancer progression. While several anti-angiogenic drugs are in use for cancer treatment, their clinical benefits are unsatisfactory. Thus, a deeper understanding of the mechanisms sustaining cancer vessel growth is fundamental to identify novel biomarkers and therapeutic targets. Alternative splicing (AS) is an essential modifier of human proteome diversity. Nevertheless, AS contribution to tumor vasculature development is poorly known. The Neuro-Oncological Ventral Antigen 2 (NOVA2) is a critical AS regulator of angiogenesis and vascular development. NOVA2 is upregulated in tumor endothelial cells (ECs) of different cancers, thus representing a potential driver of tumor blood vessel aberrancies. Here, we identified novel AS transcripts generated upon NOVA2 upregulation in ECs, suggesting a pervasive role of NOVA2 in vascular biology. In addition, we report that NOVA2 is also upregulated in ECs of gastric cancer (GC), and its expression correlates with poor overall survival of GC patients. Finally, we found that the AS of the Rap Guanine Nucleotide Exchange Factor 6 (RapGEF6), a newly identified NOVA2 target, is altered in GC patients and associated with NOVA2 expression, tumor angiogenesis, and poor patient outcome. Our findings provide a better understanding of GC biology and suggest that AS might be exploited to identify novel biomarkers and therapeutics for anti-angiogenic GC treatments.

Complexity and regulation of age-dependent alternative splicing in Brachypodium distachyon.

Alternative splicing (AS) is a gene regulatory mechanism that generates multiple transcripts of the same gene precursor by the spliceosome complex, promoting messenger RNA complexity, and proteome diversity. Although AS is extensively studied in response to environmental stresses, whether it mediates age-dependent development and how it is adjusted by growth transitions are largely unknown. Here, we comprehensively explored the AS landscape at different developmental stages in the grass model plant Brachypodium (Brachypodium distachyon). We identified abundant coding genes and noncoding transcripts subject to dynamic AS regulation during juvenile, adult, and reproductive transitions. Moreover, we revealed that SC35-LIKE SPLICING FACTOR 33 (SCL33), a serine/arginine-rich splicing factor in spliceosomes, plays a redundant and antagonistic role with its putative paralog, SCL33L, in regulating intron assembly across distinct developmental stages. In addition, we determined global AS variations in microRNA156 (miR156)-overproducing plants, in which growth transitions are delayed, and found that SPLs were regulated by miR156 in intron retention alteration in addition to mRNA clearance and translation inhibition manners. Finally, we demonstrated a complex regulatory process of age-dependent AS events in B. distachyon that was coincidently or separately regulated by miR156 and SCL33/SCL33L. These results illustrate a substantial machinery of AS that mediates phase transitions in plants.

Alternative splicing analysis benchmark with DICAST.

Alternative splicing is a major contributor to transcriptome and proteome diversity in health and disease. A plethora of tools have been developed for studying alternative splicing in RNA-seq data. Previous benchmarks focused on isoform quantification and mapping. They neglected event detection tools, which arguably provide the most detailed insights into the alternative splicing process. DICAST offers a modular and extensible framework for analysing alternative splicing integrating eleven splice-aware mapping and eight event detection tools. We benchmark all tools extensively on simulated as well as whole blood RNA-seq data. STAR and HISAT2 demonstrated the best balance between performance and run time. The performance of event detection tools varies widely with no tool outperforming all others. DICAST allows researchers to employ a consensus approach to consider the most successful tools jointly for robust event detection. Furthermore, we propose the first reporting standard to unify existing formats and to guide future tool development.

OpenCustomDB: Integration of Unannotated Open Reading Frames and Genetic Variants to Generate More Comprehensive Customized Protein Databases.

Proteomic diversity in biological samples can be characterized by mass spectrometry (MS)-based proteomics using customized protein databases generated from sets of transcripts previously detected by RNA-seq. This diversity has only been increased by the recent discovery that many translated alternative open reading frames rest unannotated at unsuspected locations of mRNAs and ncRNAs. These novel protein products, termed alternative proteins, have been left out of all previous custom database generation tools. Consequently, genetic variations that impact alternative open reading frames and variant peptides from their translated proteins are not detectable with current computational workflows. To fill this gap, we present OpenCustomDB, a bioinformatics tool that uses sample-specific RNaseq data to identify genomic variants in canonical and alternative open reading frames, allowing for more than one coding region per transcript. In a test reanalysis of a cohort of 16 patients with acute myeloid leukemia, 5666 peptides from alternative proteins were detected, including 201 variant peptides. We also observed that a significant fraction of peptide-spectrum matches previously assigned to peptides from canonical proteins got better scores when reassigned to peptides from alternative proteins. Custom protein libraries that include sample-specific sequence variations of all possible open reading frames are promising contributions to the development of proteomics and precision medicine. The raw and processed proteomics data presented in this study can be found in PRIDE repository with accession number PXD029240.

Dysfunctional gene splicing in glucose metabolism may contribute to alzheimer's disease.

The glucose metabolism is crucial for sustained brain activity as it provides energy and is a carbon source for multiple biomacromolecules; glucose metabolism decreases dramatically in Alzheimer's disease (AD) and may be a fundamental cause for its development. Recent studies reveal that the alternative splicing events of certain genes effectively regulate several processes in glucose metabolism including Insulin receptor, Insulin-degrading enzyme, Pyruvate kinase M, Receptor for Advanced Glycation Endproducts, and others, thereby, influencing glucose uptake, glycolysis, and advanced glycation end-products-mediated signaling pathways. Indeed, the discovery of aberrant alternative splicing that changes the proteomic diversity and protein activity in glucose metabolism has been pivotal in our understanding of AD development. In this review, we summarize the alternative splicing events of the glucose metabolism-related genes in AD pathology and highlight the crucial regulatory roles of splicing factors in the alternative splicing process. We also discuss the emerging therapeutic approaches for targeting splicing factors for AD treatment.

FungiProteomeDB: a database for the molecular weight and isoelectric points of the fungal proteomes.

Proteins' molecular weight (MW) and isoelectric point (pI) are crucial for their subcellular localization and subsequent function. These are also useful in 2D gel electrophoresis, liquid chromatography-mass spectrometry and X-ray protein crystallography. Moreover, visualizations like a virtual 2D proteome map of pI vs. MW are worthwhile to discuss the proteome diversity among different species. Although the genome sequence data of the fungi kingdom improved enormously, the proteomic details have been poorly elaborated. Therefore, we have calculated the MW and pI of the fungi proteins and reported them in, FungiProteomeDB, an online database (DB) https://vision4research.com/fungidb/. We analyzed the proteome of 685 fungal species that contain 7 127 141 protein sequences. The DB provides an easy-to-use and efficient interface for various search options, summary statistics and virtual 2D proteome map visualizations. The MW and pI of a protein can be obtained by searching the name of a protein, a keyword or a list of accession numbers. It also allows querying protein sequences. The DB will be helpful in hypothesis formulation and in various biotechnological applications. Database URL https://vision4research.com/fungidb/.

Role of Heterogeneous Nuclear Ribonucleoproteins in the Cancer-Immune Landscape

Cancer remains the second leading cause of death, accounting for approximately 20% of all fatalities. Evolving cancer cells and a dysregulated immune system create complex tumor environments that fuel tumor growth, metastasis, and resistance. Over the past decades, significant progress in deciphering cancer cell behavior and recognizing the immune system as a hallmark of tumorigenesis has been achieved. However, the underlying mechanisms controlling the evolving cancer-immune landscape remain mostly unexplored. Heterogeneous nuclear ribonuclear proteins (hnRNP), a highly conserved family of RNA-binding proteins, have vital roles in critical cellular processes, including transcription, post-transcriptional modifications, and translation. Dysregulation of hnRNP is a critical contributor to cancer development and resistance. HnRNP contribute to the diversity of tumor and immune-associated aberrant proteomes by controlling alternative splicing and translation. They can also promote cancer-associated gene expression by regulating transcription factors, binding to DNA directly, or promoting chromatin remodeling. HnRNP are emerging as newly recognized mRNA readers. Here, we review the roles of hnRNP as regulators of the cancer-immune landscape. Dissecting the molecular functions of hnRNP will provide a better understanding of cancer-immune biology and will impact the development of new approaches to control and treat cancer.

Alternative Polyadenylation Is a Novel Strategy for the Regulation of Gene Expression in Response to Stresses in Plants.

Precursor message RNA requires processing to generate mature RNA. Cleavage and polyadenylation at the 3'-end in the maturation of mRNA is one of key processing steps in eukaryotes. The polyadenylation (poly(A)) tail of mRNA is an essential feature that is required to mediate its nuclear export, stability, translation efficiency, and subcellular localization. Most genes have at least two mRNA isoforms via alternative splicing (AS) or alternative polyadenylation (APA), which increases the diversity of transcriptome and proteome. However, most previous studies have focused on the role of alternative splicing on the regulation of gene expression. In this review, we summarize the recent advances concerning APA in the regulation of gene expression and in response to stresses in plants. We also discuss the mechanisms for the regulation of APA for plants in the adaptation to stress responses, and suggest that APA is a novel strategy for the adaptation to environmental changes and response to stresses in plants.

SWATH-MS-based proteogenomic analysis reveals the involvement of alternative splicing in poplar upon lead stress.

Alternative splicing (AS) regulates gene expression and increases proteomic diversity for the fine tuning of stress responses in plants, but the exact mechanism through which AS functions in plant stress responses is not thoroughly understood. Here, we investigated how AS functions in poplar (Populus trichocarpa), a popular plant for bioremediation, in response to lead (Pb) stress. Using a proteogenomic analysis, we determine that Pb stress induced alterations in AS patterns that are characterized by an increased use of nonconventional splice sites and a higher abundance of Pb-responsive splicing factors (SFs) associated with Pb-responsive transcription factors. A strong Pb(II)-inducible chaperone protein, PtHSP70, that undergoes AS was further characterized. Overexpression of its two spliced isoforms, PtHSP70-AS1 and PtHSP70-AS2, in poplar and Arabidopsis significantly enhances the tolerance to Pb. Further characterization shows that both isoforms can directly bind to Pb(II), and PtHSP70-AS2 exhibits 10-fold higher binding capacities and a greater increase in expression under Pb stress, thereby reducing cellular toxicity through Pb(II) extrusion and conferring Pb tolerance. AS of PtHSP70 is found to be regulated by PtU1-70K, a Pb(II)-inducible core SF involved in 5'-splice site recognition. Because the same splicing pattern is also found in HSP70 orthologs in other plant species, AS of HSP70 may be a common regulatory mechanism to cope with Pb(II) toxicity. Overall, we have revealed a novel post-transcriptional machinery that mediates heavy metal tolerance in diverse plant species. Our findings offer new molecular targets and bioengineering strategies for phytoremediation and provide new insight for future directions in AS research.

Can blood proteome diversity among fish species help explain perfluoroalkyl acid trophodynamics in aquatic food webs?

Per- and polyfluoroalkyl substances (PFAS) are a diverse family of industrially significant synthetic chemicals infamous for extreme environmental persistence and global environmental distribution. Many PFAS are bioaccumulative and biologically active mainly due to their tendency to bind with various proteins. These protein interactions are important in determining the accumulation potential and tissue distribution of individual PFAS. Trophodynamics studies including aquatic food webs present inconsistent evidence for PFAS biomagnification. This study strives to identify whether the observed variability in PFAS bioaccumulation potential among species could correspond with interspecies protein composition differences. Specifically, this work compares the perfluorooctane sulfonate (PFOS) serum protein binding potential and the tissue distribution of ten perfluoroalkyl acids (PFAAs) detected in alewife (Alosa pseudoharengus), deepwater sculpin (Myoxocephalus thompsonii), and lake trout (Salvelinus namaycush) of the Lake Ontario aquatic piscivorous food web. These three fish sera and fetal bovine reference serum all had unique total serum protein concentrations. Serum protein-PFOS binding experiments showed divergent patterns between fetal bovine serum and fish sera, suggesting potentially two different PFOS binding mechanisms. To identify interspecies differences in PFAS-binding serum proteins, fish sera were pre-equilibrated with PFOS, fractionated by serial molecular weight cut-off filter fractionation, followed by liquid chromatography–tandem mass spectrometry analysis of the tryptic protein digests and the PFOS extracts of each fraction. This workflow identified similar serum proteins for all fish species. However, serum albumin was only identified in lake trout, suggesting apolipoproteins are likely the primary PFAA transporters in alewife and deepwater sculpin sera. PFAA tissue distribution analysis provided supporting evidence for interspecies variations in lipid transport and storage, which may also contribute to the varied PFAA accumulation in these species. Proteomics data are available via ProteomeXchange with identifier PXD039145.

Aberrant splicing in human cancer: An RNA structural code point of view.

Alternative splicing represents an essential process that occurs widely in eukaryotes. In humans, most genes undergo alternative splicing to ensure transcriptome and proteome diversity reflecting their functional complexity. Over the last decade, aberrantly spliced transcripts due to mutations in cis- or trans-acting splicing regulators have been tightly associated with cancer development, largely drawing scientific attention. Although a plethora of single proteins, ribonucleoproteins, complexed RNAs, and short RNA sequences have emerged as nodal contributors to the splicing cascade, the role of RNA secondary structures in warranting splicing fidelity has been underestimated. Recent studies have leveraged the establishment of novel high-throughput methodologies and bioinformatic tools to shed light on an additional layer of splicing regulation in the context of RNA structural elements. This short review focuses on the most recent available data on splicing mechanism regulation on the basis of RNA secondary structure, emphasizing the importance of the complex RNA G-quadruplex structures (rG4s), and other specific RNA motifs identified as splicing silencers or enhancers. Moreover, it intends to provide knowledge on newly established techniques that allow the identification of RNA structural elements and highlight the potential to develop new RNA-oriented therapeutic strategies against cancer.

Effect of Processing Methods of Human Saliva on the Proteomic Profile and Protein-Mediated Biological Processes.

The use of saliva as a protein source prior to microbiological and biological assays requires previous processing. However, the effect of these processing methods on the proteomic profile of saliva has not been tested. Stimulated human saliva was collected from eight healthy volunteers. Non-processed saliva was compared with 0.22 μm filtered, 0.45 μm filtered, and pasteurized saliva, by liquid chromatography-mass spectrometry. Data are available via ProteomeXchange with identifier PXD039248. The effect of processed saliva on microbial adhesion was tested using bacterial and fungus species and in biological cell behavior using HaCaT immortalized human keratinocytes. Two hundred and seventy-eight proteins were identified in non-processed saliva, of which 54 proteins (≈19%) were exclusive. Saliva processing reduced identified proteins to 222 (≈80%) for the 0.22 μm group, 219 (≈79%) for the 0.45 μm group, and 201 (≈72%) for the pasteurized saliva, compared to non-processed saliva. The proteomic profile showed similar molecular functions and biological processes. The different saliva processing methods did not alter microbial adhesion (ANOVA, p > 0.05). Interestingly, pasteurized saliva reduced keratinocyte cell viability. Saliva processing methods tested reduced the proteomic profile diversity of saliva but maintained similar molecular functions and biological processes, not interfering with microbial adhesion and cell viability, except for pasteurization, which reduced cell viability.

Intron dynamics reveal principles of gene regulation during the maternal-to-zygotic transition.

The maternal-to-zygotic transition (MZT) is a conserved embryonic process in animals where developmental control shifts from the maternal to zygotic genome. A key step in this transition is zygotic transcription, and deciphering the MZT requires classifying newly transcribed genes. However, due to current technological limitations, this starting point remains a challenge for studying many species. Here, we present an alternative approach that characterizes transcriptome changes based solely on RNA-seq data. By combining intron-mapping reads and transcript-level quantification, we characterized transcriptome dynamics during the Drosophila melanogaster MZT. Our approach provides an accessible platform to investigate transcriptome dynamics that can be applied to the MZT in nonmodel organisms. In addition to classifying zygotically transcribed genes, our analysis revealed that over 300 genes express different maternal and zygotic transcript isoforms due to alternative splicing, polyadenylation, and promoter usage. The vast majority of these zygotic isoforms have the potential to be subject to different regulatory control, and over two-thirds encode different proteins. Thus, our analysis reveals an additional layer of regulation during the MZT, where new zygotic transcripts can generate additional proteome diversity.

Re-evaluating the impact of alternative RNA splicing on proteomic diversity.

Alternative splicing (AS) constitutes a mechanism by which protein-coding genes and long non-coding RNA (lncRNA) genes produce more than a single mature transcript. From plants to humans, AS is a powerful process that increases transcriptome complexity. Importantly, splice variants produced from AS can potentially encode for distinct protein isoforms which can lose or gain specific domains and, hence, differ in their functional properties. Advances in proteomics have shown that the proteome is indeed diverse due to the presence of numerous protein isoforms. For the past decades, with the help of advanced high-throughput technologies, numerous alternatively spliced transcripts have been identified. However, the low detection rate of protein isoforms in proteomic studies raised debatable questions on whether AS contributes to proteomic diversity and on how many AS events are really functional. We propose here to assess and discuss the impact of AS on proteomic complexity in the light of the technological progress, updated genome annotation, and current scientific knowledge.