Identification and characterisation of sPEPs in Cryptococcus neoformans.

Short open reading frame (sORF)-encoded peptides (sPEPs) have been found across a wide range of genomic locations in a variety of species. To date, their identification, validation, and characterisation in the human fungal pathogen Cryptococcus neoformans has been limited due to a lack of standardised protocols. We have developed an enrichment process that enables sPEP detection within a protein sample from this polysaccharide-encapsulated yeast, and implemented proteogenomics to provide insights into the validity of predicted and hypothetical sORFs annotated in the C. neoformans genome. Novel sORFs were discovered within the 5' and 3' UTRs of known transcripts as well as in "non-coding" RNAs. One novel candidate, dubbed NPB1, that resided in an RNA annotated as "non-coding", was chosen for characterisation. Through the creation of both specific point mutations and a full deletion allele, the function of the new sPEP, Npb1, was shown to resemble that of the bacterial trans-translation protein SmpB.

Widespread Exaptation of L1 Transposons for Transcription Factor Binding in Breast Cancer.

L1 transposons occupy 17% of the human genome and are widely exapted for the regulation of human genes, particularly in breast cancer, where we have previously shown abundant cancer-specific transcription factor (TF) binding sites within the L1PA2 subfamily. In the current study, we performed a comprehensive analysis of TF binding activities in primate-specific L1 subfamilies and identified pervasive exaptation events amongst these evolutionarily related L1 transposons. By motif scanning, we predicted diverse and abundant TF binding potentials within the L1 transposons. We confirmed substantial TF binding activities in the L1 subfamilies using TF binding sites consolidated from an extensive collection of publicly available ChIP-seq datasets. Young L1 subfamilies (L1HS, L1PA2 and L1PA3) contributed abundant TF binding sites in MCF7 cells, primarily via their 5' UTR. This is expected as the L1 5' UTR hosts cis-regulatory elements that are crucial for L1 replication and mobilisation. Interestingly, the ancient L1 subfamilies, where 5' truncation was common, displayed comparable TF binding capacity through their 3' ends, suggesting an alternative exaptation mechanism in L1 transposons that was previously unnoticed. Overall, primate-specific L1 transposons were extensively exapted for TF binding in MCF7 breast cancer cells and are likely prominent genetic players modulating breast cancer transcriptional regulation.

Emerging evidence for functional peptides encoded by short open reading frames.

Short open reading frames (sORFs) are a common feature of all genomes, but their coding potential has mostly been disregarded, partly because of the difficulty in determining whether these sequences are translated. Recent innovations in computing, proteomics and high-throughput analyses of translation start sites have begun to address this challenge and have identified hundreds of putative coding sORFs. The translation of some of these has been confirmed, although the contribution of their peptide products to cellular functions remains largely unknown. This Review examines this hitherto overlooked component of the proteome and considers potential roles for sORF-encoded peptides.

CFTR mRNA expression is regulated by an upstream open reading frame and RNA secondary structure in its 5' untranslated region.

Post-transcriptional regulation of gene expression through 5' untranslated region (5'UTR)-encoded cis-acting elements is an important mechanism for the control of protein expression levels. Through controlling specific aspects of translation initiation, expression can be tightly regulated while remaining responsive to cellular requirements. With respect to cystic fibrosis (CF), the overexpression of cystic fibrosis transmembrane conductance regulator (CFTR) protein trafficking mutants, such as delta-F508, is of great biological and clinical interest. By understanding the post-transcriptional mechanisms that regulate CFTR expression, new procedures can be developed to enhance CFTR expression in homozygous delta-F508 CF patients. We have identified the key elements of a complex negative regulatory mechanism that is encoded within the human CFTR 5'UTR and show how these elements act in combination to restrict CFTR gene expression to a consistently low level in a transcript-specific manner. This study shows, for the first time, that endogenous human CFTR expression is post-transcriptionally regulated through a 5'UTR-mediated mechanism. We show that the very low levels of endogenous CFTR expression, compared with other low expression genes, are maintained through the co-operative inhibitory effects of an upstream open reading frame and a thermodynamically stable RNA secondary structure.

uPEPperoni: an online tool for upstream open reading frame location and analysis of transcript conservation.

BACKGROUND: Several small open reading frames located within the 5' untranslated regions of mRNAs have recently been shown to be translated. In humans, about 50% of mRNAs contain at least one upstream open reading frame representing a large resource of coding potential. We propose that some upstream open reading frames encode peptides that are functional and contribute to proteome complexity in humans and other organisms. We use the term uPEPs to describe peptides encoded by upstream open reading frames. RESULTS: We have developed an online tool, termed uPEPperoni, to facilitate the identification of putative bioactive peptides. uPEPperoni detects conserved upstream open reading frames in eukaryotic transcripts by comparing query nucleotide sequences against mRNA sequences within the NCBI RefSeq database. The algorithm first locates the main coding sequence and then searches for open reading frames 5' to the main start codon which are subsequently analysed for conservation. uPEPperoni also determines the substitution frequency for both the upstream open reading frames and the main coding sequence. In addition, the uPEPperoni tool produces sequence identity heatmaps which allow rapid visual inspection of conserved regions in paired mRNAs. CONCLUSIONS: uPEPperoni features user-nominated settings including, nucleotide match/mismatch, gap penalties, Ka/Ks ratios and output mode. The heatmap output shows levels of identity between any two sequences and provides easy recognition of conserved regions. Furthermore, this web tool allows comparison of evolutionary pressures acting on the upstream open reading frame against other regions of the mRNA. Additionally, the heatmap web applet can also be used to visualise the degree of conservation in any pair of sequences. uPEPperoni is freely available on an interactive web server at http://upep-scmb.biosci.uq.edu.au.

Differential subcellular distributions and trafficking functions of hnRNP A2/B1 spliceoforms.

Trafficking of mRNA molecules from the nucleus to distal processes in neural cells is mediated by heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 trans-acting factors. Although hnRNP A2/B1 is alternatively spliced to generate four isoforms, most functional studies have not distinguished between these isoforms. Here, we show, using isoform-specific antibodies and isoform-specific green fluorescent protein (GFP)-fusion expression constructs, that A2b is the predominant cytoplasmic isoform in neural cells, suggesting that it may play a key role in mRNA trafficking. The differential subcellular distribution patterns of the individual isoforms are determined by the presence or absence of alternative exons that also affect their dynamic behavior in different cellular compartments, as measured by fluorescence correlation spectroscopy. Expression of A2b is also differentially regulated with age, species and cellular development. Furthermore, coinjection of isoform-specific antibodies and labeled RNA into live oligodendrocytes shows that the assembly of RNA granules is impaired by blockade of A2b function. These findings suggest that neural cells modulate mRNA trafficking by regulating alternative splicing of hnRNP A2/B1 and controlling expression levels of A2b, which may be the predominant mediator of cytoplasmic-trafficking functions. These findings highlight the importance of considering isoform-specific functions for alternatively spliced proteins.

Complex evolutionary relationships among four classes of modular RNA-binding splicing regulators in eukaryotes: the hnRNP, SR, ELAV-like and CELF proteins.

Alternative RNA splicing in multicellular organisms is regulated by a large group of proteins of mainly unknown origin. To predict the functions of these proteins, classification of their domains at the sequence and structural level is necessary. We have focused on four groups of splicing regulators, the heterogeneous nuclear ribonucleoprotein (hnRNP), serine-arginine (SR), embryonic lethal, abnormal vision (ELAV)-like, and CUG-BP and ETR-like factor (CELF) proteins, that show increasing diversity among metazoa. Sequence and phylogenetic analyses were used to obtain a broader understanding of their evolutionary relationships. Surprisingly, when we characterised sequence similarities across full-length sequences and conserved domains of ten metazoan species, we found some hnRNPs were more closely related to SR, ELAV-like and CELF proteins than to other hnRNPs. Phylogenetic analyses and the distribution of the RRM domains suggest that these proteins diversified before the last common ancestor of the metazoans studied here through domain acquisition and duplication to create genes of mixed evolutionary origin. We propose that these proteins were derived independently rather than through the expansion of a single protein family. Our results highlight inconsistencies in the current classification system for these regulators, which does not adequately reflect their evolutionary relationships, and suggests that a domain-based classification scheme may have more utility.

Enzymatic recognition of 2'-modified ribonucleoside 5'-triphosphates: towards the evolution of versatile aptamers.

The quest for effective, selective and nontoxic nucleic-acid-based drugs has led to designing modifications of naturally occurring nucleosides. A number of modified nucleic acids have been made in the past decades in the hope that they would prove useful in target-validation studies and therapeutic applications involving antisense, RNAi, aptamer, and ribozyme-based technologies. Since their invention in the early 1990s, aptamers have emerged as a very promising class of therapeutics, with one drug entering the market for the treatment of age-related macular degeneration. To combat the limitations of aptamers containing naturally occurring nucleotides, chemically modified nucleotides have to be used. In order to apply modified nucleotides in aptamer drug development, their enzyme-recognition capabilities must be understood. For this purpose, several modified nucleoside 5'-triphosphates were synthesized and investigated as substrates for various enzymes. Herein, we review studies on the enzyme-recognition of various 2'-sugar-modified NTPs that were carried out with a view to their effective utilization in SELEX processes to generate versatile aptamers.

Functional implications of the emergence of alternative splicing in hnRNP A/B transcripts.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) A/B are a family of RNA-binding proteins that participate in various aspects of nucleic acid metabolism, including mRNA trafficking, telomere maintenance, and splicing. They are both regulators and targets of alternative splicing, and the patterns of alternative splicing of their transcripts have diverged between paralogs and between orthologs in different species. Surprisingly, the extent of this splicing variation and its implications for post-transcriptional regulation have remained largely unexplored. Here, we conducted a detailed analysis of hnRNP A/B sequences and expression patterns across six vertebrates. Alternative exons emerged via the introduction of new splice sites, changes in the strengths of existing splice sites, and the accumulation of auxiliary splicing regulatory motifs. Observed isoform expression patterns could be attributed to the frequency and strength of cis-elements. We found a trend toward increased splicing variation in mammals and identified novel alternatively spliced isoforms in human and chicken. Pulldown and translational assays demonstrated that the inclusion of alternative exons altered the affinity of hnRNP A/B proteins for their cognate nucleic acids and modified protein expression levels. As the hnRNPs A/B regulate several key steps in mRNA processing, the involvement of diverse hnRNP isoforms in multiple cellular contexts and species implies concomitant differences in the transcriptional output of these systems. We conclude that the emergence of alternative splicing in the hnRNPs A/B has contributed to the diversification of their roles in the regulation of alternative splicing and has thus added an unexpected layer of regulatory complexity to transcription in vertebrates.

Integrated transcription factor profiling with transcriptome analysis identifies L1PA2 transposons as global regulatory modulators in a breast cancer model.

While transposons are generally silenced in somatic tissues, many transposons escape epigenetic repression in epithelial cancers, become transcriptionally active and contribute to the regulation of human gene expression. We have developed a bioinformatic pipeline for the integrated analysis of transcription factor binding and transcriptomic data to identify transposon-derived promoters that are activated in specific diseases and developmental states. We applied this pipeline to a breast cancer model, and found that the L1PA2 transposon subfamily contributes abundant regulatory sequences to co-ordinated transcriptional regulation in breast cancer. Transcription factor profiling demonstrates that over 27% of L1PA2 transposons harbour co-localised binding sites of functionally interacting, cancer-associated transcription factors in MCF7 cells, a cell line used to model breast cancer. Transcriptomic analysis reveals that L1PA2 transposons also contribute transcription start sites to up-regulated transcripts in MCF7 cells, including some transcripts with established oncogenic properties. In addition, we verified the utility of our pipeline on other transposon subfamilies, as well as on leukemia and lung carcinoma cell lines. We demonstrate that the normally quiescent regulatory activities of transposons can be activated and alter the cancer transcriptome. In particular, the L1PA2 subfamily contributes abundant regulatory sequences, and likely plays a global role in modulating breast cancer transcriptional regulation. Understanding the regulatory impact of L1PA2 on breast cancer genomes provides additional insights into cancer genome regulation, and may provide novel biomarkers for disease diagnosis, prognosis and therapy.

Identification and functional analysis of novel BRCA1 transcripts, including mouse Brca1-Iris and human pseudo-BRCA1.

Recent characterization of the mammalian transcriptome has confirmed its predicted complexity, with many loci encoding multiple splice variants and pseudogenes. The breast cancer susceptibility gene BRCA1 is a tumour suppressor gene that produces multiple functional transcripts. For example, BRCA1-IRIS is a splice variant of BRCA1, which encodes a protein that is functionally distinct from BRCA1. Here we describe the identification of ten novel Brca1 splice variants including Brca1-Iris, the mouse orthologue of human BRCA1-IRIS. We show that Brca1-Iris is differentially expressed during mammary epithelial differentiation and regulates survival of mammary epithelial cells. Another transcript, Brca1-Delta22, expressed in both mouse and human cells, was found to be defective in transcriptional activation capacity. Finally, we show that the human BRCA1 pseudogene produces a spliced pseudoBRCA1 transcript. The identification of these transcripts has implications for the understanding of the role of BRCA1 in biology and disease and for the interpretation of mouse knockout models.

Differential subnuclear localisation of hnRNPs A/B is dependent on transcription and cell cycle stage.

The heterogeneous nuclear ribonucleoproteins A1, A2/B1 and A3 (hnRNPs A/B) are involved in many nuclear functions that are confined to distinct regions within the nucleus. To characterise and compare the distribution of the hnRNPs A/B in these subnuclear compartments, their colocalisation with spliceosomal components, nascent transcripts and other nuclear markers in HeLa cells was investigated by immunostaining and transfection of GFP constructs. The mechanisms of this localisation were further explored by treating cells with detergent, nucleases and transcription inhibitors. We have also examined the dynamics of A2/B1 throughout the cell cycle. Our results show that hnRNPs A/B have different subnuclear localisations, with A1 differentially localised to the nuclear envelope, and A2/B1 and A3 enriched around nucleoli. This pattern of distribution was dependent on RNA integrity and active transcription. The hnRNPs A/B preferentially colocalised with a subset of splicing factors. Significantly, only rarely did transcription factories colocalise with high levels of these hnRNPs. Moreover, localisation of A2/B1 changed with cell cycle stage. Our findings show that the subnuclear localisation of the hnRNPs A/B is differentially, spatially and temporally regulated, and suggest that this localisation may be relevant to their nuclear functions.

Downstream targets of heterogeneous nuclear ribonucleoprotein A2 mediate cell proliferation.

Over-expression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is regarded as an early marker for several cancers. This protein is associated with proto-oncogenes and tumor suppressor genes and has itself been described as a proto-oncogene. Our earlier experiments drew a connection between hnRNP A2/B1 levels and cell proliferation and raised the possibility that this protein contributes to the uncontrolled cell division that characterizes cancer. Limited knowledge of the downstream targets of hnRNP A2/B1 has, however, precluded a clear understanding of their roles in cancer cell growth. To define the pathways in which this protein acts we have now carried out microarray experiments with total RNA from Colo16 epithelial cells transfected with an shRNA that markedly suppresses hnRNP A2/B1 expression. The microarray data identified 123 genes, among 22 283 human gene probe sets, with altered expression levels in hnRNP A2/B1-depleted cells. Ontological analysis showed that many of these downstream targets are involved in regulation of the cell cycle and cell proliferation and that this group of proteins is significantly over-represented amongst the affected proteins. The changes detected in the microarray experiments were confirmed by real-time PCR for a subset of proliferation-related genes. Immunoprecipitation-RT-PCR demonstrated that hnRNP A2/B1 formed complexes with the transcripts of many of the verified downstream genes, suggesting that hnRNP A2/B1 contributes to the regulation of these genes. These results reinforce the conclusion that hnRNP A2/B1 is associated with cellular processes that affect the cell cycle and proliferation.

Targeted disruption of Brca1 in restricted compartments of the mouse mammary epithelia.

Tumours arising in BRCA1 mutation carriers have a characteristic phenotype, the molecular and cellular basis of which is unknown. To address the hypothesis that this phenotype reflects a role for BRCA1 in either in the basal or the stem cell compartments of the mammary epithelia, we have targeted its disruption to K14 and K6a expressing cells of the mouse. Unlike MMTV and WAP driven conditional knockout models of Brca1, these two models did not result in any observable changes in the mammary gland. Our results suggest that BRCA1-associated tumours arise either in K14 and K6a negative basal cells of the mammary gland, or possibly from transdifferentiation of luminal epithelia.

Evidence for conservation and selection of upstream open reading frames suggests probable encoding of bioactive peptides.

BACKGROUND: Approximately 40% of mammalian mRNA sequences contain AUG trinucleotides upstream of the main coding sequence, with a quarter of these AUGs demarcating open reading frames of 20 or more codons. In order to investigate whether these open reading frames may encode functional peptides, we have carried out a comparative genomic analysis of human and mouse mRNA 'untranslated regions' using sequences from the RefSeq mRNA sequence database. RESULTS: We have identified over 200 upstream open reading frames which are strongly conserved between the human and mouse genomes. Consensus sequences associated with efficient initiation of translation are overrepresented at the AUG trinucleotides of these upstream open reading frames, while comparative analysis of their DNA and putative peptide sequences shows evidence of purifying selection. CONCLUSION: The occurrence of a large number of conserved upstream open reading frames, in association with features consistent with protein translation, strongly suggests evolutionary maintenance of the coding sequence and indicates probable functional expression of the peptides encoded within these upstream open reading frames.

5'-untranslated regions with multiple upstream AUG codons can support low-level translation via leaky scanning and reinitiation.

Upstream AUGs (uAUGs) and upstream open reading frames (uORFs) are common features of mRNAs that encode regulatory proteins and have been shown to profoundly influence translation of the main ORF. In this study, we employed a series of artificial 5'-untranslated regions (5'-UTRs) containing one or more uAUGs/uORFs to systematically assess translation initiation at the main AUG by leaky scanning and reinitiation mechanisms. Constructs containing either one or two uAUGs in varying contexts but without an in-frame stop codon upstream of the main AUG were used to analyse the leaky scanning mechanism. This analysis largely confirmed the ranking of different AUG contextual sequences that was determined previously by Kozak. In addition, this ranking was the same for both the first and second uAUGs, although the magnitude of initiation efficiency differed. Moreover, approximately 10% of ribosomes exhibited leaky scanning at uAUGs in the most favourable context and initiated at a downstream AUG. A second group of constructs containing different numbers of uORFs, each with optimal uAUGs, were used to measure the capacity for reinitiation. We found significant levels of initiation at the main ORF even in constructs containing four uORFs, with nearly 10% of ribosomes capable of reinitiating five times. This study shows that for mRNAs containing multiple uORFs/uAUGs, ribosome reinitiation and leaky scanning are efficient mechanisms for initiation at their main AUGs.

Characterization of mouse profilaggrin: evidence for nuclear engulfment and translocation of the profilaggrin B-domain during epidermal differentiation.

Filaggrin is a keratin filament associated protein that is expressed in granular layer keratinocytes and derived by sequential proteolysis from a polyprotein precursor termed profilaggrin. Depending on the species, each profilaggrin molecule contains between 10 and 20 filaggrin subunits organized as tandem repeats with a calcium-binding domain at the N- terminal end. We now report the characterization of the complete mouse gene. The structural organization of the mouse gene is identical to the human profilaggrin gene and consists of three exons with a 4 kb intron within the 5' noncoding region and a 1.7 kb intron separating the sequences encoding the calcium-binding EF-hand motifs. A processed pseudogene was found embedded within the second intron. The third and largest exon encodes the second EF-hand, a basic domain (designated the B-domain) followed by 12 filaggrin repeats and a unique C-terminal tail domain. A polyclonal antibody raised against the conceptually translated sequence of the B-domain specifically stained keratohyalin granules and colocalized with a filaggrin antibody in granular layer cells. In upper granular layer cells, B-domain containing keratohyalin granules were in close apposition to the nucleus and, in some cells, appeared to be completely engulfed by the nucleus. In transition layer cells, B-domain staining was evident in the nucleus whereas filaggrin staining remained cytoplasmic. Nuclear staining of the B-domain was also observed in primary mouse keratinocytes induced to differentiate. This study has also revealed significant sequence homology between the mouse and human promoter sequences and in the calcium-binding domain but the remainder of the protein-coding region shows substantial divergence.

Regulation of MAPK activation, AP-1 transcription factor expression and keratinocyte differentiation in wounded fetal skin.

Fetal epithelium retains the ability to re-epithelialize a wound in organotypic culture in a manner not dependent on the presence of underlying dermal substrata. This capacity is lost late in the third trimester of gestation or after embryonic day 17 (E(17)) in the rat such that embryonic day 19 (E(19)) wounds do not re-epithelialize. Moreover, wounds created in E(17) fetuses in utero heal in a regenerative, scar-free fashion. To investigate the molecular events regulating re-epithelialization in fetal skin, the wound-induced expression profile and tissue localization of activator protein 1 (AP-1) transcription factors c-Fos and c-Jun was characterised in E(17) and E(19) skin using organotypic fetal cultures. The involvement of mitogen-activated protein kinase (MAPK) signaling in mediating wound-induced transcription factor expression and wound re-epithelialization was assessed, with the effect of wounding on the expression of keratinocyte differentiation markers determined. Our results show that expression of AP-1 transcription factors was induced immediately by wounding and localized predominantly to the epidermis in E(17) and E(19) skin. c-fos and c-jun induction was transient in E(17) skin with MAPK-dependent c-fos expression necessary for the re-epithelialization of an excisional wound in organotypic culture. In E(19) skin, AP-1 expression persisted beyond 12 h post-wounding, and marked upregulation of the keratinocyte differentiation markers keratin 10 and loricrin was observed. No such changes in the expression of keratin 10 or loricrin occurred in E(17) skin. These findings indicate that re-epithelialization in fetal skin is regulated by wound-induced AP-1 transcription factor expression via MAPK and the differentiation status of keratinocytes.

Isolation (from a basal cell carcinoma) of a functionally distinct fibroblast-like cell type that overexpresses Ptch.

In this study we report on the isolation and characterization of a nonepithelial, nontumorigenic cell type (BCC1) derived from a basal cell carcinoma from a patient. The BCC1 cells share many characteristics with dermal fibroblasts, such as the expression of vimentin, lack of expression of cytokeratins, and insensitivity to agents that cause growth inhibition and differentiation of epithelial cells; however, significant differences between BCC1 cells and fibroblasts also exist. For example, BCC1 cells are stimulated to undergo DNA synthesis in response to interferon-gamma, whereas dermal fibroblasts are not. More over, BCC1 cells overexpress the basal cell carcinoma-specific genes ptch and ptch2. These data indicate that basal cell carcinomas are associated with a functionally distinct population of fibroblast-like cells that overexpress known tumor-specific markers (ptch and ptch2).

Characterization of the mouse hnRNP A2/B1/B0 gene and identification of processed pseudogenes.

The mouse hnRNP A2/B1/B0 gene has been cloned using a PCR-based strategy and sequenced. Analysis of this sequence showed that the gene organization closely follows that of the human orthologue with 12 exons and 11 introns. The hnRNP A2/B1/B0 gene gives rise to four splice variants through alternative splicing of exons 2 and 9. RT-PCR assays indicated that all splice variants were expressed in mouse brain, skin, and stomach tissues of varying ages, although their ratios to one another varied with age and tissue type. We also identified a small subset of all polyadenylated splice variants that included intron 11, which shows 94% sequence identity between human and mouse. Several processed pseudogenes were identified in the mouse genome. A search of the mouse genome databases located five pseudogenes, four of which are presumed to be non-functional because of the presence of premature stop codons, large deletions or rearrangements within the coding region. The fifth, which possesses putative promoter elements and has a coding sequence identical to that of the hnRNP A2 mRNA variant, may be functional.