Encoding Protein Isoforms Research Articles

CLN3 transcript complexity revealed by long-read RNA sequencing analysis

BackgroundBatten disease is a group of rare inherited neurodegenerative diseases. Juvenile CLN3 disease is the most prevalent type, and the most common pathogenic variant shared by most patients is the “1-kb” deletion which removes two internal coding exons (7 and 8) in CLN3. Previously, we identified two transcripts in patient fibroblasts homozygous for the 1-kb deletion: the ‘major’ and ‘minor’ transcripts. To understand the full variety of disease transcripts and their role in disease pathogenesis, it is necessary to first investigate CLN3 transcription in “healthy” samples without juvenile CLN3 disease.MethodsWe leveraged PacBio long-read RNA sequencing datasets from ENCODE to investigate the full range of CLN3 transcripts across various tissues and cell types in human control samples. Then we sought to validate their existence using data from different sources.ResultsWe found that a readthrough gene affects the quantification and annotation of CLN3. After taking this into account, we detected over 100 novel CLN3 transcripts, with no dominantly expressed CLN3 transcript. The most abundant transcript has median usage of 42.9%. Surprisingly, the known disease-associated ‘major’ transcripts are detected. Together, they have median usage of 1.5% across 22 samples. Furthermore, we identified 48 CLN3 ORFs, of which 26 are novel. The predominant ORF that encodes the canonical CLN3 protein isoform has median usage of 66.7%, meaning around one-third of CLN3 transcripts encode protein isoforms with different stretches of amino acids. The same ORFs could be found with alternative UTRs. Moreover, we were able to validate the translational potential of certain transcripts using public mass spectrometry data.ConclusionOverall, these findings provide valuable insights into the complexity of CLN3 transcription, highlighting the importance of studying both canonical and non-canonical CLN3 protein isoforms as well as the regulatory role of UTRs to fully comprehend the regulation and function(s) of CLN3. This knowledge is essential for investigating the impact of the 1-kb deletion and rare pathogenic variants on CLN3 transcription and disease pathogenesis.

Unraveling novel mRNA transcripts of the human DNA N-glycosylase 1 (NTHL1) gene with the implementation of an innovative targeted DNA-seq assay

The human NTHL1 gene encodes a DNA glycosylase that plays a key role in the base excision repair (BER) pathway, repairing oxidative DNA damage and maintaining genome integrity. The physiological activity of NTHL1 is crucial in preventing genetic alterations that can lead to cancer. In this study, we employed an innovative targeted DNA sequencing (DNA-seq) methodology to explore the transcriptional landscape of the NTHL1 gene, revealing previously uncharacterized alternative splicing events and novel exons. Our designed approach provided significantly improved sequencing depth and coverage, enabling the identification of novel NTHL1 mRNA transcripts. Bioinformatics analysis confirmed all annotated splice junctions of the main NTHL1 transcripts (v.1 – v.3) and revealed novel mRNA transcripts (NTHL1 v.4 – v.9) derived from splicing events between annotated exons as well as mRNAs containing previously uncharacterized exons (NTHL1 v.10 – v.14). Quantitative PCR analysis highlighted a diverse expression pattern of these novel transcripts across different human cell lines, suggesting cell-specific roles and regulatory mechanisms. Notably, NTHL1 v.5 was overexpressed in luminal A breast cancer cells (MCF-7), while v.13 was prominent in triple negative (BT-20), HER2 + breast cancer (SK-BR-3), prostate, colorectal cancer cells and HEK-293 cells. Our findings suggest that specific novel NTHL1 transcripts may encode protein isoforms with distinct structural features, as indicated by ribosome profiling datasets, while others containing premature termination codons could function as long non-coding RNAs. These insights enhance our understanding of NTHL1 regulatory role and its potential as a biomarker and therapeutic target in human malignancies. This study underscores the importance of exploring the transcriptional diversity of NTHL1 to fully elucidate its role in cancer pathobiology.

Microsatellite instability at U2AF-binding polypyrimidic tract sites perturbs alternative splicing during colorectal cancer initiation

BackgroundMicrosatellite instability (MSI) due to mismatch repair deficiency (dMMR) is common in colorectal cancer (CRC). These cancers are associated with somatic coding events, but the noncoding pathophysiological impact of this genomic instability is yet poorly understood. Here, we perform an analysis of coding and noncoding MSI events at the different steps of colorectal tumorigenesis using whole exome sequencing and search for associated splicing events via RNA sequencing at the bulk-tumor and single-cell levels.ResultsOur results demonstrate that MSI leads to hundreds of noncoding DNA mutations, notably at polypyrimidine U2AF RNA-binding sites which are endowed with cis-activity in splicing, while higher frequency of exon skipping events are observed in the mRNAs of MSI compared to non-MSI CRC. At the DNA level, these noncoding MSI mutations occur very early prior to cell transformation in the dMMR colonic crypt, accounting for only a fraction of the exon skipping in MSI CRC. At the RNA level, the aberrant exon skipping signature is likely to impair colonic cell differentiation in MSI CRC affecting the expression of alternative exons encoding protein isoforms governing cell fate, while also targeting constitutive exons, making dMMR cells immunogenic in early stage before the onset of coding mutations. This signature is characterized by its similarity to the oncogenic U2AF1-S34F splicing mutation observed in several other non-MSI cancer.ConclusionsOverall, these findings provide evidence that a very early RNA splicing signature partly driven by MSI impairs cell differentiation and promotes MSI CRC initiation, far before coding mutations which accumulate later during MSI tumorigenesis.

ApoE Cascade Hypothesis in the pathogenesis of Alzheimer's disease and related dementias.

The ε4 allele of the apolipoprotein E gene (APOE4) is a strong genetic risk factor for Alzheimer's disease (AD) and several other neurodegenerative conditions, including Lewy body dementia (LBD). The three APOE alleles encode protein isoforms that differ from one another only at amino acid positions 112 and 158: apoE2 (C112, C158), apoE3 (C112, R158), and apoE4 (R112, R158). Despite progress, it remains unclear how these small amino acid differences in apoE sequence among the three isoforms lead to profound effects on aging and disease-related pathways. Here, we propose a novel "ApoE Cascade Hypothesis" in AD and age-related cognitive decline, which states that the biochemical and biophysical properties of apoE impact a cascade of events at the cellular and systems levels, ultimately impacting aging-related pathogenic conditions including AD. As such, apoE-targeted therapeutic interventions are predicted to be more effective by addressing the biochemical phase of the cascade.

Genome-Wide Analysis of Alternative Splicing in Zea mays during Maize Iranian Mosaic Virus Infection

Maize Iranian mosaic virus (MIMV) infects several gramineous plants and is an economically important nucleorhabdovirus in Iran. Maize responds to MIMV infection at the transcriptional level. Alternative splicing (AS) is a mechanism that generates multiple mRNAs from a single pre-mRNA, often encoding protein isoforms with functional differences. We carried out genome-wide analysis of AS responses to MIMV in maize seedlings and identified genes involved in this molecular response. The AS events we investigated included skipped exons, alternative 3′ splice site, alternative 5′ splice site, mutually exclusive exons, and retained introns. In total 10,881 maize genes showed AS, of which 601 genes were involved in response to MIMV-infection and 186 were found only in uninfected maize. AS was identified in some of the genes that are involved in disease resistance or pathogenicity pathways. We demonstrated that in MIMV-infects maize, host genes that are involved in symptom development, virus multiplication, resistance to pathogens and host-pathogen interaction are affected by AS mechanism. Gene network analysis showed that ten genes represent the hubs for the protein network in maize and that they are involved in response to pathogen attack and include 26S proteasome, 14–3-3-like protein A, Rop family, mitogen-activated protein kinase, ubiquitin and serine/threonine-protein kinases. In conclusion, we showed that AS occurs as a transcriptional regulatory mechanism in maize response to MIMV infection and we identified genes that have the key roles in pathogenicity pathways that were differentially spliced in infected seedlings.

A novel exon generates ubiquitously expressed alternatively spliced new transcript of mouse Abcc4 gene

Abcc4 gene codes for a protein (ABCC4) involved in the transportation of different classes of drugs outside the cells. Various important drugs transported by ABCC4 include antiviral and anticancer drugs as well as endogenous molecules such as bile acids, cyclic nucleotides, folates, prostaglandins and steroids. Alternative splicing generates multiple mRNAs that encode protein isoforms having diverse functions. In this study, we have identified a novel transcript of mouse Abcc4 gene using a combination of bioinformatics and molecular biology techniques. This transcript was found to be different from the reported transcript in having a different first exon that was found to be located on previously identified first intron. Newly identified transcript was found to be expressed across different tissues we studied and in different developmental stages. Expression level of novel and reported transcripts was studied using quantitative real-time PCR. After conceptually translating the novel transcript, various post-translational modifications were studied. Translation efficiency and predicted half life of encoded protein isoforms were analysed in silico. Molecular modelling was performed to compare the structural differences in both isoforms. The diversity at N-termini in these protein isoforms explains the diverse function of ABCC4 in mouse.

A Comparison of Resources for the Annotation of a De Novo Assembled Transcriptome in the Molting Gland (Y-Organ) of the Blackback Land Crab, Gecarcinus lateralis.

Next-generation sequencing technologies are revolutionizing crustacean biology. De novo assembly of RNA sequencing (RNA-seq) data allows researchers to catalog and quantify genes expressed in tissues of a species that lacks a complete genome sequence. RNA-seq has become an important tool for understanding phenotypic plasticity and the responses of organisms to environmental cues. However, there are challenges with identification of assembled contiguous sequences (contigs) without a reference genome. Thus, the selection of resources for annotating contigs is critical for the downstream analysis of gene functions. A de novo-assembled transcriptome of the Gecarcinus lateralis molting gland, or Y-organ (YO), was used to compare two functional annotation packages, Trinotate and Blast2GO. The assembled transcriptome contained 229,278 contigs derived from YOs from animals in intermolt, premolt (early, mid, and late), and postmolt stages. Gene identification using BLAST against four databases and functional annotation using Gene Ontologies were conducted. The analysis revealed two major limitations of de novo assembly: (1) assembly using Trinity generates redundant contigs and (2) transcripts that encode protein isoforms are not distinguished with current computational tools. It is recommended that the NCBI Non-Redundant, SwissProt, TrEMBL, and Uniref90 databases be used to maximize gene identification. Trinotate is preferred for assigning functions to identified genes, as the package uses multiple databases for annotation. The differences between packages to generate Gene Ontology (GO) terms are attributed to the databases used for inputs: Trinotate uses both Pfam and BLAST databases, while Blast2GO uses only the BLAST database. InterProScan was used to verify the GO terms assigned via BLAST. A comprehensive annotation of de novo assembled transcriptome is necessary for the downstream analysis of differentially expressed transcripts in the YO over the molt cycle.

Abstract 827: Polyphenon E treatment affects p73 mRNA levels

Abstract Prostate cancer (PCa) is the most common malignancy among men in the USA. Polyphenon E (PolyE) is a standardized blend of polyphenols found in green tea extract, which has been shown to have chemoprevention value in PCa models, but the molecular mechanism(s) have not been elucidated. The p73 gene is a member of the p53 tumor suppressor family. A p73 dinucleotide polymorphism (DNP) (rs1801173) is a G4C14-to-A4T14 linked pair of transitions located in exon 2 and lies between the P1 and P2 gene promoters. The P1 and P2 p73 gene promoters are the transcription initiation sites for mRNAs encoding TAp73 and deltaNp73 (DNp73) isoforms, respectively. These p73 mRNA isoforms, TAp73 and áNp73, encode protein isoforms which differ in their N-termini. TAp73 isoforms include the full-length N-terminal sequence and are transcriptionally active. áNp73 isoforms lack the N-terminal trans-activation domain and are dominate negative. Recently, we reported the p73 DNP allele was associated with (1) decreased risk [OR = 0.55, 95%CI = 0.31-0.99] for aggressive PCa and (2) increased TAp73/DNp73 protein isoform ratios in ten human cancer cell lines. We hypothesize that exposure to PolyE induces changes in TAp73/DNp73 mRNA isoform ratios. Our goal for this study is to assess the effect of PolyE on total p73 mRNA levels and TAp73/DNp73 mRNA isoform ratios using two human PCa cell lines with different p73 DNP genotypes: DU145 (wild-type) and PC-3 (heterozygous). Cultured cells were treated with PolyE (100 or 200 mg/L). Cellular RNA was isolated, converted to cDNA and used in TaqMan RT-PCR assays to detect total p73, TAp73 or DNp73 isoform mRNAs. Our data from PolyE treatments of both DU145 and PC-3 cells are consistent with increased p73 mRNA levels (2.05 and 1.60 fold, respectively) and higher TAp73/DNp73 mRNA ratios (1.13 and 1.29 fold, respectively) at the low Poly E level (100 mg/L). At the high PolyE level (200 mg/L), the TAp73/DNp73 mRNA ratios significantly decreased for both DU145 and PC-3 (0.54 and 0.49 respectively). However, the overall p73 mRNA levels was not changed in DU145 (0.91 fold), but increased in PC-3 (1.60 fold) as compared with level in negative control at the high Poly E concentration. Therefore, in this exploratory study, PolyE treatment of the two PCa cell lines tested using a more moderate concentration of 100 mg/L results in higher overall p73 levels and increased TAp73/DNp73 mRNA ratios. This data could provide a potential molecular rationale for the observation that Poly E treatments provide chemopreventative impact against PCa. Citation Format: L. Michael Carastro, Ricardo A. Cordova, Christopher D. Cole, Jong Y. Park. Polyphenon E treatment affects p73 mRNA levels. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 827.

Abstract 3003: p73 DNP genotype influences effects of etoposide and 5-aza-2’-deoxycytidine on p73 mRNA isoforms ratios in human cancer cell lines

Abstract Background: The p73 gene is a member of the p53 tumor suppressor family. A dinucleotide polymorphism (DNP) in p73 is a G4C14-to-A4T14 linked pair of transitions located in exon 2, between the P1 and P2 promoters, from which TAp73 and DeltaNp73 (DNp73) mRNA isoforms are transcribed, respectively. A p73 dinucleotide polymorphism (DNP) (rs1801173) is a G4C14-to-A4T14 linked pair of transitions located in exon 2 and lies between the P1 and P2 gene promoters. The P1 and P2 p73 gene promoters are the transcription initiation sites for mRNAs encoding TAp73 and deltaNp73 (DNp73) isoforms, respectively. These p73 mRNA isoforms, TAp73 and αNp73, encode protein isoforms which differ in their N-termini. TAp73 isoforms include the full-length N-terminal sequence and are transcriptionally active. αNp73 isoforms lack the N-terminal trans-activation domain and are dominate negative. Recently, we reported the p73 DNP allele was associated with (1) decreased risk [OR = 0.55, 95%CI = 0.31-0.99] for aggressive prostate cancer and (2) increased TAp73/DNp73 protein isoform ratios in ten human cancer cell lines. Etoposide is known to induce p73 gene expression. Others recently reported treatment of T-47D cells with 5-aza-2’-deoxycytidine (5AZA) resulted in increased TAp73/DNp73 mRNA ratios. Hypothesis: We hypothesized that treatment with 5AZA increases the TAp73/DNp73 mRNA ratios in human cell lines and that the presence of the p73 DNP allele potentiates 5AZA-induced increases TAp73/DNp73 cellular ratios. Methods. We treated human cancer cell lines with 50 microM etoposide for 24 hrs and/or 20 microM 5AZA for 48 hrs. RNA was isolated, converted to cDNA and used in TaqMan RT-PCR assays to detect total p73, TAp73 or αNp73 mRNAs. Results: Cell lines wild-type for p73 DNP (DU145, T47-D, MDA-MB-468) had TAp73/DNp73 mRNA ratios that were lower than those heterozygous for p73 DNP (PC-3 and MDA-MB-231) following co-treatment with 5AZA and etoposide or with etoposide alone (p<0.01 for DU145 & T47-D). PC-3 and MDA-MB-231 cell lines had TAp73/DNp73 mRNA ratios following 5AZA treatment (0.8546 and 0.7138, respectively) that were equal to or greater than the three cell lines wild type for p73 DNP: DU145, T47-D and MDA-MB-468 (0.8626, 0.6480, 0.3866, respectively). Conclusion: Of the five cell lines used in this study, cells that harbor a p73 DNP allele had the highest TAp73/DNp73 mRNA ratios following co-treatment with 5AZA and etoposide or with etoposide alone as compared to wild-type cell p73 DNP, which is consistent with our previously reported data. Lastly, our data in this work assessing the effect of 5AZA treatment of T47-D cells on TAp73/DNp73 mRNA ratios is not consistent with a previously reported study. Citation Format: Zachary Connelly, L. Michael Carastro. p73 DNP genotype influences effects of etoposide and 5-aza-2’-deoxycytidine on p73 mRNA isoforms ratios in human cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3003.

Differential Splicing Alters Subcellular Localization of the Alpha but not Beta Isoform of the MIER1 Transcriptional Regulator in Breast Cancer Cells

MIER1 was originally identified in a screen for novel fibroblast growth factor activated early response genes. The mier1 gene gives rise to multiple transcripts encoding protein isoforms that differ in their amino (N-) and carboxy (C-) termini. Much of the work to date has focused on the two C-terminal variants, MIER1α and β, both of which have been shown to function as transcriptional repressors. Our previous work revealed a dramatic shift in MIER1α subcellular localization from nuclear in normal breast tissue to cytoplasmic in invasive breast carcinoma, suggesting that loss of nuclear MIER1α may play a role in breast cancer development. In the present study, we investigated whether alternative splicing to include a cassette exon and produce an N–terminal variant of MIER1α affects its subcellular localization in MCF7 breast carcinoma cells. We demonstrate that this cassette exon, exon 3A, encodes a consensus leucine-rich nuclear export signal (NES). Inclusion of this exon in MIER1α to produce the MIER1-3Aα isoform altered its subcellular distribution in MCF7 cells from 81% nuclear to 2% nuclear and this change in localization was abrogated by mutation of critical leucines within the NES. Treatment with leptomycin B (LMB), an inhibitor of the nuclear export receptor CRM1, resulted in a significant increase in the percentage of cells with nuclear MIER1-3Aα, from 4% to 53%, demonstrating that cytoplasmic localization of this isoform was due to CRM1-dependent nuclear export. Inclusion of exon 3A in MIER1β to produce the N-terminal variant MIER1-3Aβ however had little effect on the nuclear targeting of this isoform. Our results demonstrate that alternative splicing to include exon 3A specifically affects the localization pattern of the α isoform.

Systematic analysis of protein pools, isoforms, and modifications affecting turnover and subcellular localization.

In higher eukaryotes many genes encode protein isoforms whose properties and biological roles are often poorly characterized. Here we describe systematic approaches for detection of either distinct isoforms, or separate pools of the same isoform, with differential biological properties. Using information from ion intensities we have estimated protein abundance levels and using rates of change in stable isotope labeling with amino acids in cell culture isotope ratios we measured turnover rates and subcellular distribution for the HeLa cell proteome. Protein isoforms were detected using three data analysis strategies that evaluate differences between stable isotope labeling with amino acids in cell culture isotope ratios for specific groups of peptides within the total set of peptides assigned to a protein. The candidate approach compares stable isotope labeling with amino acids in cell culture isotope ratios for predicted isoform-specific peptides, with ratio values for peptides shared by all the isoforms. The rule of thirds approach compares the mean isotope ratio values for all peptides in each of three equal segments along the linear length of the protein, assessing differences between segment values. The three in a row approach compares mean isotope ratio values for each sequential group of three adjacent peptides, assessing differences with the mean value for all peptides assigned to the protein. Protein isoforms were also detected and their properties evaluated by fractionating cell extracts on one-dimensional SDS-PAGE prior to trypsin digestion and MS analysis and independently evaluating isotope ratio values for the same peptides isolated from different gel slices. The effect of protein phosphorylation on turnover rates was analyzed by comparing mean turnover values calculated for all peptides assigned to a protein, either including, or excluding, values for cognate phosphopeptides. Collectively, these experimental and analytical approaches provide a framework for expanding the functional annotation of the genome.

Splicing Mechanisms That Generate Distinct Isoforms of Protein 4.1R During Terminal Erythroid Differentiation.

Abstract 4036Poster Board III-972The protein 4.1R gene is a large complex gene with two translation initiation sites (AUG1 and AUG2) that encode protein isoforms with distinct N-terminal structure and function. Expression of these isoforms is regulated by alternative splicing at either of two splice acceptor sites that flank exon 2' (E2'), in which AUG1 is located. In late erythroblasts E2' is excluded, ensuring translation at AUG2 and synthesis of 80kDa protein 4.1R isoforms. Our earlier studies (EMBO J. 27:122-31, 2008) described a two-step intrasplicing pathway that enforces this splicing outcome exclusively in 4.1R pre-mRNA initiated at exon 1A (E1A), the major transcription start site in late erythropoiesis. The downstream exon 1B (E1B) first splices to the proximal acceptor site at E2' to generate an intermediate structure, which is then re-processed by splicing of E1A to the internal acceptor at E2, removing E1B as well as E2'/AUG1. Experiments with minigenes suggested that intrasplicing is likely independent of specific promoter elements at E1A, but absolutely requires 5' splice site and branch point motifs associated with E1B. Here we sought evidence for functionality of these latter elements in the more physiological context of the endogenous 4.1R gene. The intrasplicing model predicts that morpholino oligonucleotides complementary to key regulatory motifs will block the first step of the pathway in natural 4.1R pre-mRNA transcripts, and yield inappropriate splicing of E1A to the first acceptor at E2'. Antisense morpholinos directed against the E1B branchpoint or E1B 5'splice site were transfected into cells and 4.1R splicing was examined 48hrs later by RT-PCR analysis. Both anti-4.1 morpholinos, but not a control morpholino, resulted in a concentration-dependent shift of E1A splicing to the proximal E2' acceptor site. In other studies we explored whether intrasplicing could occur internally within a gene, using a model pre-mRNA in which a constitutive exon was engineered between E1A and E1B. Analysis of this experiment suggested that any exon upstream of active E1B would follow the intrasplicing pathway and delete E2'. We speculate that internal E1B-like elements in other genes could be selectively activated or silenced by splicing regulatory motifs in order to control downstream splice acceptor choice. Preliminary experiments indicate that weakening the E1B 5' splice site and its upstream pyrimidine tract might permit such regulation as observed for other alternative exons. Together these results provide additional support for the intrasplicing model and suggest that it could function more widely in human genes to coordinate splicing events and generate multiple protein isoforms with distinct functions. Disclosures:No relevant conflicts of interest to declare.

Centrosomin: A Complex Mix of Long and Short Isoforms Is Required for Centrosome Function During Early Development in Drosophila melanogaster

Centrosomin (Cnn) is a required core component in mitotic centrosomes during syncytial development and the presence of Cnn at centrosomes has become synonymous with fully functional centrosomes in Drosophila melanogaster. Previous studies of Cnn have attributed this embryonic function to a single isoform or splice variant. In this study, we present new evidence that significantly increases the complexity of cnn. Rather than a single isoform, Cnn function can be attributed to two unique classes of proteins that comprise a total of at least 10 encoded protein isoforms. We present the initial characterization of a new class of Cnn short isoforms required for centrosome function during gametogenesis and embryogenesis. We also introduce new evidence for a complex mix of Cnn isoforms present during early embryogenesis. Finally, we reexamine cnn mutations, in light of the short isoforms, and find previously overlooked differences attributable to allele-specific mutant phenotypes. This study addresses several questions surrounding Cnn function at the centrosome during embryogenesis and shows that cnn function cannot be ascribed to a single protein.

Splice variants of the human ZC3H14 gene generate multiple isoforms of a zinc finger polyadenosine RNA binding protein

The human ZC3H14 gene encodes an evolutionarily conserved Cys 3His zinc finger protein that binds specifically to polyadenosine RNA and is thus postulated to modulate post-transcriptional gene expression. Expressed sequence tag (EST) data predicts multiple splice variants of both human and mouse ZC3H14. Analysis of ZC3H14 expression in both human cell lines and mouse tissues confirms the presence of multiple alternatively spliced transcripts. Although all of these transcripts encode protein isoforms that contain the conserved C-terminal zinc finger domain, suggesting that they could all bind to polyadenosine RNA, they differ in other functionally important domains. Most of the alternative transcripts encode closely related proteins (termed isoforms 1, 2, 3, and 3short) that differ primarily in the inclusion of three small exons, 9, 10, and 11, resulting in predicted protein isoforms ranging from 82 to 64 kDa. Each of these closely related isoforms contains predicted classical nuclear localization signals (cNLS) within exons 7 and 11. Consistent with the presence of these putative nuclear targeting signals, these ZC3H14 isoforms are all localized to the nucleus. In contrast, an additional transcript encodes a smaller protein (34 kDa) with an alternative first exon (isoform 4). Consistent with the absence of the predicted cNLS motifs located in exons 7 and 11, ZC3H14 isoform 4 is localized to the cytoplasm. Both EST data and experimental data suggest that this variant is enriched in testes and brain. Using an antibody that detects endogenous ZC3H14 isoforms 1–3 reveals localization of these isoforms to nuclear speckles. These speckles co-localize with the splicing factor, SC35, suggesting a role for nuclear ZC3H14 in mRNA processing. Taken together, these results demonstrate that multiple transcripts encoding several ZC3H14 isoforms exist in vivo. Both nuclear and cytoplasmic ZC3H14 isoforms could have distinct effects on gene expression mediated by the common Cys 3His zinc finger polyadenosine RNA binding domain.

Splice-mediated Variants of Proteins (SpliVaP) – data and characterization of changes in signatures among protein isoforms due to alternative splicing

BackgroundIt is often the case that mammalian genes are alternatively spliced; the resulting alternate transcripts often encode protein isoforms that differ in amino acid sequences. Changes among the protein isoforms can alter the cellular properties of proteins. The effect can range from a subtle modulation to a complete loss of function.Results(i) We examined human splice-mediated protein isoforms (as extracted from a manually curated data set, and from a computationally predicted data set) for differences in the annotation for protein signatures (Pfam domains and PRINTS fingerprints) and we characterized the differences & their effects on protein functionalities. An important question addressed relates to the extent of protein isoforms that may lack any known function in the cell. (ii) We present a database that reports differences in protein signatures among human splice-mediated protein isoform sequences.Conclusion(i) Characterization: The work points to distinct sets of alternatively spliced genes with varying degrees of annotation for the splice-mediated protein isoforms. Protein molecular functions seen to be often affected are those that relate to: binding, catalytic, transcription regulation, structural molecule, transporter, motor, and antioxidant; and the processes that are often affected are nucleic acid binding, signal transduction, and protein-protein interactions. Signatures are often included/excluded and truncated in length among protein isoforms; truncation is seen as the predominant type of change. Analysis points to the following novel aspects: (a) Analysis using data from the manually curated Vega indicates that one in 8.9 genes can lead to a protein isoform of no "known" function; and one in 18 expressed protein isoforms can be such an "orphan" isoform; the corresponding numbers as seen with computationally predicted ASD data set are: one in 4.9 genes and one in 9.8 isoforms. (b) When swapping of signatures occurs, it is often between those of same functional classifications. (c) Pfam domains can occur in varying lengths, and PRINTS fingerprints can occur with varying number of constituent motifs among isoforms – since such a variation is seen in large number of genes, it could be a general mechanism to modulate protein function. (ii) Data: The reported resource (at ) provides the community ability to access data on splice-mediated protein isoforms (with value-added annotation such as association with diseases) through changes in protein signatures.

Restricted expression of the human DAZ protein in premeiotic germ cells

The role of the Y chromosome-encoded Deleted in Azoospermia (DAZ) gene family in spermatogenesis remains unclear. The ability of men without the DAZ gene to produce sperm, as well as the lack of selective pressure on DAZ exon sequences during evolution, casts doubts on its functional significance. Most men have four DAZ genes encoding protein isoforms that differ significantly in size. However, published western blots showed only a single "DAZ" band, raising the possibility that not all four DAZ genes are expressed. RT-PCR, western blotting and immunostaining were used to study the expression of the four DAZ genes and the autosomal DAZL gene in human testes and in tissue culture cells. RNA transcripts of all four DAZ genes were found in the testis, but at much lower levels than that of the DAZL transcripts. Expression in cultured somatic cells showed that DAZ transcripts encoding multiple DAZ repeats were translated inefficiently. No DAZ proteins could be unambiguously identified on western blots when the testicular samples from three patients without the DAZ genes were used as negative controls. Nonetheless, low levels of DAZ were detected in the cytoplasm of spermatogonia by immunostaining. The expression of DAZ proteins in adult human testes is restricted to the spermatogonia and suggests a premeiotic role.

Identification and analysis of human RCAN3 ( DSCR1L2) mRNA and protein isoforms

Human RCAN3 (Regulator of calcineurin 3; previously known as DSCR1L2, Down syndrome critical region gene 1-like 2) is a five-exon gene mapped on chromosome 1 and belongs to the human RCAN gene family which also includes RCAN1 and RCAN2. The novel denomination RCAN for genes and proteins, instead of DSCR1L (Down syndrome critical region gene 1-like) has recently been widely discussed. The aim of the present work was to perform a multiple approach analysis of five RCAN3 mRNA and encoded protein isoforms, two of which have been identified for the first time in this research. The two new RCAN3 mRNA isoforms, RCAN3-2,4,5, which lacks exon 3, and RCAN3-2,3,5, which lacks exon 4, were identified during RCAN3 RT-PCR (reverse transcription-polymerase chain reaction) cloning, the product of which unexpectedly revealed the presence of five isoforms as opposed to the three previously known. In order to analyze the expression pattern of the five RCAN3 mRNA isoforms in seven different human tissues, a quantitative relative RT-PCR was performed: interestingly, all isoforms are present in all tissues investigated, with a statistically significant constant prevalence of RCAN3 isoform (the most complete, “reference” isoform). The RCAN3 locus expression level was comparable in all seven tissues analyzed, considering all isoforms, which indicates a ubiquitous expression of this human RCAN family member. To date two possible interactors have been described for this protein: human cardiac troponin I (TNNI3) and calcineurin. Here we report the interaction between the new RCAN3 variants and TNNI3, demonstrated by both yeast cotransformation and by the GST (glutathione-sepharose transferase) fusion protein assay, as was to be expected from the presence of exon 2 whose product has been seen to be sufficient for binding to TNNI3.

A Bpag1 isoform involved in cytoskeletal organization surrounding the nucleus

Bpag1/dystonin proteins are giant cytoskeletal interacting proteins postulated to cross-link cytoskeletal filaments and thereby maintain cellular integrity. Loss of function of non-epithelial Bpag1 isoforms results in neuromuscular dysfunction and early postnatal death in mice. Multiple Bpag1 transcripts have been described, including those encoding protein isoforms that vary at the N-terminal end. Here, we have analyzed the subcellular localizations and cytoskeletal interactions of two isoforms, termed Bpag1a1 and Bpag1a2. We demonstrate that novel sequence at the 5' end of the Bpag1a2 transcript codes for an N-terminal transmembrane domain and targets the protein to the perinuclear region of the cell. Furthermore, we show that the endogenous Bpag1a2 protein is also present in the perinuclear region in myoblast cells. Differences in Bpag1a1 and Bpag1a2 with respect to the extent of their interactions with microtubules and microfilaments are also described, with Bpag1a2 fusion protein serving largely to associate with microfilaments surrounding the nucleus and Golgi apparatus. The overall structure and subcellular localizations of Bpag1a2 indicate possible functions in nuclear envelope structuring, nuclear tethering, and organization of membranous structures surrounding the nucleus.

Organization of the mouse Ruk locus and expression of isoforms in mouse tissues

Ruk is a recently identified gene with a complex pattern of expression in mammalian cells and tissues. Multiple Ruk transcripts and several protein isoforms have been detected in various types of cells. Ruk proteins have multidomain organization characteristic of adapter proteins involved in regulation of signal transduction. Interaction of some Ruk isoforms with several signalling proteins, including the p85 regulatory subunit of the Class IA PI 3-kinase, c-Cbl and Grb2, has been demonstrated. Ruk l, an isoform with three SH3 domains, inhibits lipid kinase activity of the PI 3-kinase in vitro; overexpression of this protein induces apoptotic cell death of primary neurons in culture and changes in membrane trafficking in other cultured cells. However, shorter isoforms of Ruk block pro-apoptotic effect of Ruk l, suggesting that expression of different combinations of Ruk proteins in cells could be involved in the regulation of their survival and other intracellular processes. To understand the mechanism of differential expression of Ruk proteins we studied organization of the mouse Ruk gene and its transcripts. Twenty-four exons of the Ruk gene span over 320 kb of the mouse chromosome X. Analysis of cDNA clones, ESTs and products of RT-PCR amplifications with different combinations of primers revealed how alternative splicing and promoter usage generate a variety of Ruk transcripts and encoded protein isoforms in different mouse tissues.

Identification and Characterization of the Human Protein Kinase-like Gene NTKL: Mitosis-Specific Centrosomal Localization of an Alternatively Spliced Isoform

Although the centrosome has an essential role in mitosis, its molecular components have not been fully elucidated. Here, we describe the molecular cloning and characterization of the human gene NTKL, which encodes an evolutionarily conserved kinase-like protein. NTKL mRNA is found ubiquitously in human tissues. NTKL is located on 11q13 and is mapped around chromosomal breakpoints found in several carcinomas, suggesting that NTKL dysfunction may be involved in carcinogenesis. Alternative splicing generates two variant forms of NTKL mRNA that encode protein isoforms with internal deletions. When fused to green fluorescent protein, the full-length product and one of the variant proteins are found in cytoplasm. The other variant product also exists in the cytoplasm during interphase, but is found in the centrosomes during mitosis. Endogenous NTKL protein is also localized to the centrosomes during mitosis. This cell-cycle-dependent centrosomal localization suggests that NTKL is involved in centrosome-related cellular functions.