Alternative Start Codons Research Articles

SET Nuclear Oncogene Associates with Microcephalin/MCPH1 and Regulates Chromosome Condensation

Primary microcephaly is an autosomal recessive disorder characterized by marked reduction in human brain size. Microcephalin (MCPH1), one of the genes mutated in primary microcephaly, plays an important role in DNA damage checkpoint control and mitotic entry. Additionally, MCPH1 ensures the proper temporal activation of chromosome condensation during mitosis, by acting as a negative regulator of the condensin II complex. We previously found that deletion of the of the MCPH1 N terminus leads to the premature chromosome condensation (PCC) phenotype. In the present study, we unexpectedly observed that a truncated form of MCPH1 appears to be expressed in MCPH1(S25X/S25X) patient cells. This likely results from utilization of an alternative translational start codon, which would produce a mutant MCPH1 protein with a small deletion of its N-terminal BRCT domain. Furthermore, missense mutations in the MCPH1 cluster at its N terminus, suggesting that intact function of this BRCT protein-interaction domain is required both for coordinating chromosome condensation and human brain development. Subsequently, we identified the SET nuclear oncogene as a direct binding partner of the MCPH1 N-terminal BRCT domain. Cells with SET knockdown exhibited abnormal condensed chromosomes similar to those observed in MCPH1-deficient mouse embryonic fibroblasts. Condensin II knockdown rescued the abnormal chromosome condensation phenotype in SET-depleted cells. In addition, MCPH1 V50G/I51V missense mutations, impair binding to SET and fail to fully rescue the abnormal chromosome condensation phenotype in Mcph1(-/-) mouse embryonic fibroblasts. Collectively, our findings suggest that SET is an important regulator of chromosome condensation/decondensation and that disruption of the MCPH1-SET interaction might be important for the pathogenesis of primary microcephaly.

Alternative translation start sites are conserved in eukaryotic genomes

Alternative start AUG codons within a single transcript can contribute to diversity of the proteome; however, their functional significance remains controversial. Here, we provide comparative genomics evidence that alternative start codons are under negative selection in vertebrates, insects and yeast. In genes where the annotated start codon (sAUG) resides within the suboptimal nucleotide context, the downstream in-frame AUG codons (dAUG) among the first ∼30 codon sites are significantly more conserved between species than in genes where the sAUG resides within the optimal context. Proteomics data show that this difference is not an annotation artifact and that dAUGs are in fact under selection as alternative start sites. The key optimal, and sometimes suboptimal, context-determining nucleotides of both the sAUG and dAUGs are conserved. Selection for secondary start sites is stronger in genes with the weak primary start site. Genes with multiple conserved start sites are enriched for transcription factors, and tend to have longer 5′UTRs and higher degree of alternative splicing. Together, these results imply that the use of alternative start sites by means of leaky mRNA scanning is a functional mechanism under selection for increased efficiency of translation and/or for translation of different N-terminal protein variants.

Cryptic Epitopes Induce High-Titer Humoral Immune Response in Patients with Cancer

In search of novel markers for diagnosis, prognosis, and therapy of cancer, screening of rcDNA expression libraries with patient's sera has been established as a valuable tool for identification of cancer-specific Ags. Interestingly, besides the expected humoral responses to annotated proteins, patients with cancer were frequently found to have serum Abs that bind to peptides without homology to known proteins. So far, the nature of these unconventional epitopes and their possible significance in tumor immunology have never been thoroughly investigated. In our study, we specifically analyzed humoral immune response toward such peptides in patients with pancreatic or breast cancer using yeast-displayed cDNA expression libraries derived from tumor tissue. A detailed analysis of the identified peptides revealed that they originated from translation of sequences outside annotated open reading frames and may derive from the use of alternative start codons or from DNA indel mutations. In several cases, the corresponding mRNA templates have a known association with cancer. In a final analysis, we were able to detect one of these tumor Ags in cancer tissue arrays by a selected Fab-Ab. We conclude that cryptic epitopes may elicit specific humoral immune responses in patients with cancer and thus play a role in immunologic surveillance. Due to the high prevalence of immune responses against some of the peptides, they may also be valuable markers for cancer diagnosis, prognosis, or therapy monitoring.

Sequence variability and evolution of the terminal overlapping VP5 gene of the infectious bursal disease virus

The infectious bursal disease virus (IBDV; Birnaviridae family) constitutes one of the main threats to the poultry industry worldwide. Most of the progress in the molecular epidemiology of this virus has been achieved through the study of the coding region of the capsid protein VP2. Little research has been done regarding the molecular evolution and the epidemiological implications of genetic variability of other IBDV genome regions. In this article, the gene that codes the non-structural protein VP5 was analyzed. Although this protein is not essential for the virus replication, recent evidence indicates that it could be related to the virulent phenotype and the adaptive capacity of the virus. The VP5 gene is also of evolutionary interest because it has an open reading frame that terminally overlaps with the pVP2-VP4-VP3 polyprotein coding region. In the first part of this study, the full VP5 gene of a South American strain was characterized. The results revealed that the VP5 gene of Uruguayan hypervirulent IBDV strains (vvIBDV) lacks the alternative AUG start codon characteristic of the vvIBDV strains that have been described to date. Instead, as occurs in classic and variant strains, this VP5 gene has an AUG start site located four codons downstream and, consequently, it codes for a 145 amino acid long protein rather than the putative 149 amino acid long protein of other vvIBDV. In spite of this, these viruses conserved the VP5 and VP2 amino acid signature of the hypervirulent strains and clustered with reference vvIBDV sequences. This finding may represent evidence that the VP5 gene could be evolving by changing the translation initiation site. In the second part of this study, an evolutionary analysis including the sequences reported in this study together with most of VP5 sequences available in the GenBank, showed the existence of a complex system of selective pressures controlling the evolution of the VP5 gene. Using the dN/dS index, we found a strong purifying selection exerted on the 5' terminal overlapping region of VP2 that would be constraining the evolution of VP5. These results reinforce the hypothesis that the VP5 gene was originated late in the IBDV evolution by a mechanism of genetic overprinting. The results described in this study provided new information about the dynamics of the IBDV genome and revealed some of the mechanisms at play in the evolution of this virus. Since VP5 seems to be related to viral pathogenicity, this evolutionary information might be useful to highlight the impact of the genetic variation of this protein on the epidemiology of IBDV.

Abstract 5575: Mass spectrometry analysis of the posttranslational modifications of alpha-enolase from pancreatic ductal adenocarcinoma cells

Abstract Enolase is one of the most abundantly expressed cytosolic proteins in many organisms, and it is a key glycolytic enzyme that catalyzes the dehydration of 2-phosphoglycerate to phosphoenolpyruvate. Recently, accumulating evidence revealed that, in addition to its innate glycolytic function, the well characterized old enzyme enolase is a multifunctional protein and plays an important role in several biological and pathophysiological processes: 1) by using an alternative start codon, the α-enolase mRNA can be translated into a 37 kDa protein which lacks the first 96 amino acid residues. This protein is localized in the nucleus and can bind to the c-myc P2 promoter and negatively regulate transcription of the proto-oncogene; 2) α-enolase has been detected on the surface of hematopoietic cells such as monocytes, T cells and B cells, neuronal cells and endothelial cells as a strong plasminogen-binding receptor, modulating the pericellular and intravascular fibrinolytic system. The expression of α-enolase on the surface of a variety of eukaryotic cells has been found to be dependent on the pathophysiological conditions of these cells, however, how α-enolase is displayed on the cell surface remains unknown; 3) increased expression of enolase has been reported to correlated with progression of tumors, such as neuroendocrine tumors, neuroblastoma and lung cancers, and enolase has been considered to be a diagnostic marker for many tumors. Our previous studies demonstrated that α-enolase is up-regulated at both the mRNA and protein levels in pancreatic ductal adenocarcinoma (PDAC). PDAC is the fourth leading cause of cancer-related deaths in Western countries. The mean life expectancy is 15-18 months for patients with local and regional disease, and only 3-6 months for those with metastatic disease. In this present work, we further characterized the α-enolase from PDAC by reversed-phase liquid chromatography nanospray tandem mass spectrometry (LC-MS/MS) analysis and an LTQ-Orbitrap instrument, and identified multiple posttranslational modifications of α-enolase, such as phosphorylation, acetylation, and methylation. The mass spectrometer LTQ-Orbitrap provides high accuracy mass measurement that is essential for the validation of modified peptide identifications and the reduction of false positive identifications. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5575.

Identification of a Consensus DNA Binding Sequence for nGdf5

Growth and differentiation factor 5 (Gdf5) is a member of the BMP family of secreted growth factors and is associated with joint formation and cartilage maintenance. Recently, however, a nuclear variant of Gdf5, called nGdf5, was discovered. It is produced by translation of the Gdf5 transcript from a downstream alternative start codon. A yeast one‐hybrid screen suggested that Gdf5 may interact with an enhancer element in the Col11a2 gene, suggesting the hypothesis that nGdf5 regulates transcription. CASTing (cyclic amplification and selection of targets) was performed to identify a consensus DNA binding sequence for nGdf5. CASTing involved several cycles of incubating nGdf5 with random oligonucleotides, imunoprecipitating the resulting DNA‐protein complexes, and then amplifying the selected DNA sequences by PCR. After four cycles of CASTing, the selected DNA probes were cloned, sequenced, and aligned. The sequence (T/G)GGTG(G/T)G was present in 15% of the selected and amplified probes, and another 58% contained 1‐ or 2‐bp mismatches to this consensus sequence. Electrophoretic mobility shift assays verified that nGdf5 binds specifically to (T/G)GGTG(G/T)G. These results suggest that the novel protein nGdf5 binds to DNA, either directly or in cooperation with a binding partner, and are consistent with the possibility that nGdf5 helps regulate transcription.

Nuclear Bmp4: a novel ROC1 and ROC2 binding partner

Bone morphogenetic protein 4 (Bmp4) is a well known growth factor that regulates gene expression through the SMAD signaling pathway. Bmp4 is involved in many developmental processes and has been identified as an important factor in several cancers. Due to the observation of Bmp4 in the nuclei of some colon cancer tissues by Madoz‐Gurpide et al. and the recent discovery of a nuclear variant of Bmp2 (nBmp2) in our lab, we hypothesized that a nuclear variant of Bmp4 (nBmp4) also exists. Immunocytochemistry experiments confirmed the presence of Bmp4 in the nuclei of several cell lines, and subsequent analyses determined that nBmp4 is translated from an alternative start codon downstream of the signal peptide sequence, which allows a bipartite nuclear localization signal to direct translocation of nBmp4 to the nucleus. Yeast two‐hybrid experiments were performed in order to identify potential nBmp4 binding partners. Among the identified proteins were regulator of cullins 1 and 2 (ROC1 and ROC2), which are components of SCF E3 ubiquitin ligases. The putative interactions between nBmp4 and ROC1 and ROC2 have been confirmed by co‐immunoprecipitation, and preliminary evidence suggests that nBmp4 also interacts with another member of the SCF complex, Cullin1. These interactions suggest that nBmp4 plays a role in the ubiquitin‐proteasome pathway. Research supported by NIH grant AR48839 and the BYU Cancer Research Center.

Nuclear variants of bone morphogenetic proteins

BackgroundBone morphogenetic proteins (BMPs) contribute to many different aspects of development including mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. They have previously been recognized only as secreted growth factors, but the present study detected Bmp2, Bmp4, and Gdf5/CDMP1 in the nuclei of cultured cells using immunocytochemistry and immunoblotting of nuclear extracts.ResultsIn all three proteins, a bipartite nuclear localization signal (NLS) was found to overlap the site at which the proproteins are cleaved to release the mature growth factors from the propeptides. Mutational analyses indicated that the nuclear variants of these three proteins are produced by initiating translation from downstream alternative start codons. The resulting proteins lack N-terminal signal peptides and are therefore translated in the cytoplasm rather than the endoplasmic reticulum, thus avoiding proteolytic processing in the secretory pathway. Instead, the uncleaved proteins (designated nBmp2, nBmp4, and nGdf5) containing the intact NLSs are translocated to the nucleus. Immunostaining of endogenous nBmp2 in cultured cells demonstrated that the amount of nBmp2 as well as its nuclear/cytoplasmic distribution differs between cells that are in M-phase versus other phases of the cell cycle.ConclusionsThe observation that nBmp2 localization varies throughout the cell cycle, as well as the conservation of a nuclear localization mechanism among three different BMP family members, suggests that these novel nuclear variants of BMP family proteins play an important functional role in the cell.

Viral adaptation to immune selection pressure by HLA class I–restricted CTL responses targeting epitopes in HIV frameshift sequences

CD8+ cytotoxic T lymphocyte (CTL)–mediated immune responses to HIV contribute to viral control in vivo. Epitopes encoded by alternative reading frame (ARF) peptides may be targeted by CTLs as well, but their frequency and in vivo relevance are unknown. Using host genetic (human leukocyte antigen [HLA]) and plasma viral sequence information from 765 HIV-infected subjects, we identified 64 statistically significant (q < 0.2) associations between specific HLA alleles and sequence polymorphisms in alternate reading frames of gag, pol, and nef that did not affect the regular frame protein sequence. Peptides spanning the top 20 HLA-associated imprints were used to test for ex vivo immune responses in 85 HIV-infected subjects and showed responses to 10 of these ARF peptides. The most frequent response recognized an HLA-A*03–restricted +2 frame–encoded epitope containing a unique A*03-associated polymorphism at position 6. Epitope-specific CTLs efficiently inhibited viral replication in vitro when viruses containing the wild-type sequence but not the observed polymorphism were tested. Mutating alternative internal start codons abrogated the CTL-mediated inhibition of viral replication. These data indicate that responses to ARF-encoded HIV epitopes are induced during natural infection, can contribute to viral control in vivo, and drive viral evolution on a population level.

Evolution of Relative Reading Frame Bias in Unidirectional Prokaryotic Gene Overlaps

Pairs of unidirectional (same strand) genes can overlap in one of two phases (relative reading frames). There is a striking bias in the relative abundance of prokaryotic gene overlaps in the two possible phases. A simple model is presented based on unidirectional gene overlaps evolving from nonoverlapping gene pairs, through the adoption of alternative start codons by the downstream genes. Potential alternative start codons within upstream gene sequences were found to occur at greater frequencies in one phase, corresponding to the most prevalent phase of gene overlaps. We therefore suggest that the phase bias of overlapping genes is primarily a consequence of the N-terminal extension of downstream genes through adoption of new start codons.

Techniques for accurate protein identification in shotgun proteomic studies of human, mouse, bovine, and chicken lenses

Analysis of shotgun proteomics datasets requires techniques to distinguish correct peptide identifications from incorrect identifications, such as linear discriminant functions and target/decoy protein databases. We report an efficient, flexible proteomic analysis workflow pipeline that implements these techniques to control both peptide and protein false discovery rates. We demonstrate its performance by analyzing two-dimensional liquid chromatography separations of lens proteins from human, mouse, bovine, and chicken lenses. We compared the use of International Protein Index databases to UniProt databases and no-enzyme SEQUEST searches to tryptic searches. Sequences present in the International Protein Index databases allowed detection of several novel crystallins. An alternate start codon isoform of βA4 was found in human lens. The minor crystallin γN was detected for the first time in bovine and chicken lenses. Chicken γS was identified and is the first member of the γ-crystallin family observed in avian lenses.Electronic supplementary materialThe online version of this article (doi:10.1007/s12177-009-9042-6) contains supplementary material, which is available to authorized users.

The Role of ETV6/TEL in Hematopoiesis and Lymphoid Neoplasia.

Abstract 972The transcriptional repressor TEL (Translocation Ets Leukemia) also known as ETV6 (Ets variant gene 6) located on human chromosome 12 is essential for hematopoietic stem cell (HSC) maintenance. Abnormalities involving TEL on the short arm of chromosome 12 including deletions and translocations are common in a surprisingly wide spectrum of hematological and non-hematological malignancies. Genome-wide analysis of leukemic samples revealed the loss of TEL expression and loss of heterozygousity (LOH) in more than half of B-cell ALL patients. We have previously reported on the generation of transgenic zebrafish expressing TEL-AML1 that develop pre-B ALL after long latency, and recapitulate the same phenotype and molecular events in human leukemias. In this study, we first used these transgenic fish to investigate the role of TEL in leukemia development. Our data shows that the loss of TEL expression contributes to, and accelerates the leukemic phenotype in our TEL-AML1 transgenic zebrafish. TEL is a selective regulator of lymphoid and HSC survival. The mechanism by which TEL modulates adult lymphoid and HSC renewal is yet to be elucidated. Moreover, studies of TEL expression and role in lymphoma development are lacking. At the genetic level, the highly conserved alternative start codon, and the initiation of translation at Methionine-43 of TEL creates a shorter isoform that is mostly nuclear. Utilizing specific antibodies that distinguish between the expression patterns of the two TEL isoforms, we demonstrate that the differential cytoplasmic and nuclear localization of TEL isoforms in human cells is involved in B- and T-cell lymphoid maturation. Our studies uncover a nuclear trafficking mechanism that is likely to regulate the nuclear repression and tumor suppressor functions of TEL. We next investigated the role of TEL in normal hematopoiesis, and TEL tumor suppressor functions in lymphoid malignancies in a novel model of zebrafish lymphoma. To study the role of TEL in hematopoiesis, we isolated the zebrafish TEL coding region and promoter elements from a BAC library, and found that TEL is expressed in early embryonic stages, in hematopoietic and neural lineages, and its expression colocalized with the hematopoietic stem cell (HSC) markers cMyb, Flk1 and RUNX1. In this model, the loss of TEL expression using morpholinos and dominant negative approaches in wild type zebrafish led to the development of lymphomas in 15% of these fish. This prompted us to investigate the expression of TEL in human lymphomas. Loss of TEL expression or the cytoplasmic localization of TEL isoforms in tumor cells from lymphoma patients were detected in eleven out of twenty three patients investigated so far (48%), and correlated with the non-Hodgkin diffuse large B-cell lymphomas and follicular subtypes as well as the aggressiveness of lymphomas. Flow cytometric analyses of lymphoma samples associated with loss of TEL expression demonstrated a CD19+ve, CD20+ve, CD10+ve, and CD45+ve phenotype. In cellular assays, we demonstrate that the activity and cellular localization of TEL are tightly regulated at multiple levels, and coordinated by P38 kinase activity. Our studies highlight a novel tumor suppressor role of TEL/ETV6 in lymphoma development, and establish TEL/ETV6 expression as a useful biomarker for non-Hodgkin lymphoma diagnosis and disease progression. Disclosures:No relevant conflicts of interest to declare.

TRIP8b Splice Variants Form a Family of Auxiliary Subunits that Regulate Gating and Trafficking of HCN Channels in the Brain

Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels, which generate the I(h) current, mediate a number of important brain functions. The HCN1 isoform regulates dendritic integration in cortical pyramidal neurons and provides an inhibitory constraint on both working memory in prefrontal cortex and spatial learning and memory in the hippocampus. Altered expression of HCN1 following seizures may contribute to the development of temporal lobe epilepsy. Yet the regulatory networks and pathways governing HCN channel expression and function in the brain are largely unknown. Here, we report the presence of nine alternative N-terminal splice forms of the brain-specific cytoplasmic protein TRIP8b and demonstrate the differential effects of six isoforms to downregulate or upregulate HCN1 surface expression. Furthermore, we find that all TRIP8b isoforms inhibit channel opening by shifting activation to more negative potentials. TRIP8b thus functions as an auxiliary subunit that provides a mechanism for the dynamic regulation of HCN1 channel expression and function.

Nuclear Bmp2 (nBmp2) Alters the Expression of Several Genes

Bone morphogenetic protein 2 (Bmp2) is a secreted growth factor that modulates many essential physiological and developmental processes through extracellular signaling. Bmp2 signaling is essential in neuronal differentiation, embryonic vasculogenesis, and cardiac development. Recent identification of a novel nuclear variant of Bmp2 challenged its sole classification as a secreted growth factor. Nuclear Bmp2 (nBmp2) is translated from an alternative start codon downstream of the signal peptide sequence, which allows a bipartite nuclear localization signal to direct translocation to the nucleus. In order to elucidate the function of nBmp2, microarray analyses were performed on cells transfected with nBmp2. These analyses indicated that over‐expression of nBmp2 alters the expression levels of hundreds of different genes. From this list, genes were selected for further analysis by quantitative RT‐PCR. This work confirmed that nBmp2 significantly alters the expression levels of several genes that are expressed in neuronal and cardiac tissue. These results suggest a functional role for nBmp2 in development or maintenance of neurological and circulatory systems. This work was supported by the NIH grant AR4883 and by an award from the Fulton Family Foundation to support undergraduate research.

Mice bearing a targeted inactivation of nBmp2 show decreased muscle strength

An important growth factor in the body, bone morphogenetic protein (Bmp2) contributes to mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. We have recently discovered a novel variant of this protein in the nucleus (nBmp2). This protein is produced by translation from a downstream alternative start codon and is localized to the nucleus by means of a bipartite nuclear localization signal. To determine the function of nBmp2, a mouse was engineered in which the conventional Bmp2 growth factor is still expressed normally, but the nuclear variant, nBmp2, cannot be translocated to the nucleus. The mice appear phenotypically normal at birth and are fertile. A series of flexed limb‐hang strength tests, however, revealed that mutant mice have significantly reduced strength compared to wild type mice. Western blot analysis performed on mitochondrial extracts showed increased amounts of cytochrome C in mutant compared to wild type muscle mitochondria. These results suggest that nBmp2 may affect muscle strength by playing a role in mitochondrial function. This work was supported by NIH grant AR48839 and by an award from the Fulton Family Foundation to promote undergraduate research.

Determining the function of nuclear Bmp4

Bone morphogenetic protein 4 (Bmp4) is a well known growth factor that regulates gene expression through the SMAD signaling pathway. Bmp4 is involved in many developmental processes and has been identified as an important factor in several cancers, including melanoma, ovarian cancer, and colon cancer. Surprisingly, Madoz‐Gurpide et al. (2006) observed Bmp4 in the nuclei of a minor percentage of cells in colon cancer tissues. In addition, our lab has recently discovered a nuclear variant of Bmp2 (nBmp2), the TGF‐β family member most closely related to Bmp4. These observations led us to hypothesize that a nuclear variant of Bmp4 (nBmp4) also exists. Preliminary analyses have been performed that verified nuclear localization of Bmp4 in several cancer cell lines. Subsequent analyses suggest that nBmp4 is translated from an alternative start codon downstream of the signal peptide sequence, which allows a bipartite nuclear localization signal to direct translocation of nBmp4 to the nucleus. In an effort to determine the function of nBmp4, a yeast two‐hybrid screen was performed using a cDNA library from MCF‐7 breast cancer cells. Several proteins were identified as potential interacting partners. These interacting proteins suggest a role for nBmp4 in protein degradation, reactive oxygen species modulation, and apoptosis. Research supported by NIH grant AR48839 and the Brigham Young University Cancer Research Center.

Bacterial Genome Partitioning: N-Terminal Domain of IncC Protein Encoded by Broad-Host-Range Plasmid RK2 Modulates Oligomerisation and DNA Binding

ParA Walker ATPases form part of the machinery that promotes better-than-random segregation of bacterial genomes. ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding. Unusually, and for as yet unknown reasons, parA (incC) of IncP-1 plasmids is translated from alternative start codons producing two forms, IncC1 (364 aa) and IncC2 (259 aa), whose ratio varies between hosts. IncC2 could be detected as an oligomeric form containing dimers, tetramers and octamers, but the N-terminal extension present in IncC1 favours nucleotide-stimulated dimerisation as well as high-affinity and ATP-dependent non-specific DNA binding. The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides. Mixing IncC1 and IncC2 improved polymerisation and DNA binding. Thus, the NTD may modulate the polymerisation interface, facilitating polymerisation/depolymerisation and DNA binding, to promote the cycle that drives partitioning.

A Rare Human Sequence Variant Reveals Myocardin Autoinhibition

Myocardin (MYOCD) is a transcriptional co-activator that promotes cardiac or smooth muscle gene programs through its interaction with myocyte-enhancing factor (MEF2) or serum-response factor (SRF). Isoforms of MYOCD with a truncated amino terminus show increased activity when compared with those with the full-length amino terminus, but how this is achieved remains unknown. We identified a rare human sequence variation in MYOCD in a patient with congenital heart disease that resulted in a missense mutation at codon 259 (K259R). This variation created a hypomorphic cardiac isoform with impaired SRF binding and transactivation capacity but did not impair the smooth muscle isoform of MYOCD, which lacks the amino terminus. Consistent with differential effects of the amino terminus on the K259R mutation, we found that the cardiac-specific amino terminus acted in an autoinhibitory fashion to bind MYOCD via specific negatively charged residues and thereby repressed SRF-dependent MYOCD activity. This effect was exaggerated in the MYOCD-K259R mutant. The amino terminus was sufficient to impair MYOCD-dependent fibroblast conversion into smooth muscle cells as well as cardiomyocyte hypertrophy. These findings identify a novel mechanism that regulates levels of MYOCD-dependent activation of the SRF genetic program differentially in cardiac and smooth muscle.

Evidence for Insertional RNA Editing in Humans

Large-scale analysis directly at the protein level holds the promise of uncovering features not apparent or present at the gene level [1-3]. Although mass spectrometry (MS)-based proteomics can now identify and quantify thousands of cellular proteins in large-scale proteomics experiments, much of the peptide information contained in these experiments remains unassigned [4]. Here, we use such information to discover a previously unreported mechanism creating altered protein forms. Linker histones H1 and high-mobility group (HMG) proteins are abundant nuclear proteins that regulate gene expression through modulation of chromatin structure [5-8]. In the high-resolution MS analysis of histone H1 and HMG protein fractions isolated from human cells, we discovered peptides that mapped upstream of the known translation start sites of these genes. No alternative upstream start site exists in the genome, but analysis of Expressed Sequence Tag (EST) databases revealed that these N-terminally extended (ET) proteins are due to in-frame translation of the 5' untranslated region (5'UTR) sequences of the transcripts. The new translation start sites are created by a single uridine insertion between AG, reflecting a previously unreported RNA-editing mechanism. To our knowledge, this is the first report of RNA-insertion editing in humans and may be an example of the type of discoveries possible with modern proteomics methods.

Identification of an alternative translation initiation site for the Pantoea ananatis lycopene cyclase ( crtY) gene in E. coli and its evolutionary conservation

Previous sequence analyses of the lycopene cyclase gene ( crtY) from Pantoea ananatis revealed that translation of its protein product in Escherichia coli began at the ATG start codon. We found, however, that this enzyme could also be produced in E. coli without the ATG start codon present. Results of experiments using crtY mutants revealed that a GTG (Val) sequence, located in-frame and 24 bp downstream of the ATG, could act as a potential start codon. Additionally, a point-mutated GTA (Val), replaced from alternative GTG start codon, also displayed its potential as a start codon although the strength as a translation initiation codon was considerably weak. This finding suggests that non-ATG codons, especially one base pairing with the anticodon (3′-UAC-5′) in fMet-tRNA, might be also able to function as start codon in translation process. Furthermore, amino acid sequence alignment of lycopene cyclases from different sources suggested that a Val residue located within the N-terminus of these enzymes might be used as an alternative translation initiation site. In particular, presence of a conserved Asp, located in-frame and 12 bp upstream of potential start codon, supports this assumption in view of the fact that Asp ( GAT or GAC) can function as part of the Shine–Dalgano sequence (AG GAGG).