Sequence determinants in human polyadenylation site selection

BACKGROUND: Differential polyadenylation is a widespread mechanism in higher eukaryotes producing mRNAs with different 3' ends in different contexts. This involves several alternative polyadenylation sites in the 3' UTR, each with its specific strength. Here, we analyze the vicinity of human polyadenylation signals in search of patterns that would help discriminate strong and weak polyadenylation sites, or true sites from randomly occurring signals. RESULTS: We used human genomic sequences to retrieve the region downstream of polyadenylation signals, usually absent from cDNA or mRNA databases. Analyzing 4956 EST-validated polyadenylation sites and their -300/+300 nt flanking regions, we clearly visualized the upstream (USE) and downstream (DSE) sequence elements, both characterized by U-rich (not GU-rich) segments. The presence of a USE and a DSE is the main feature distinguishing true polyadenylation sites from randomly occurring A(A/U)UAAA hexamers. While USEs are indifferently associated with strong and weak poly(A) sites, DSEs are more conspicuous near strong poly(A) sites. We then used the region encompassing the hexamer and DSE as a training set for poly(A) site identification by the ERPIN program and achieved a prediction specificity of 69 to 85% for a sensitivity of 56%. CONCLUSION: The availability of complete genomes and large EST sequence databases now permit large-scale observation of polyadenylation sites. Both U-rich sequences flanking both sides of poly(A) signals contribute to the definition of "true" sites. However, the downstream U-rich sequences may also play an enhancing role. Based on this information, poly(A) site prediction accuracy was moderately but consistently improved compared to the best previously available algorithm

Diversity of Protein and mRNA Forms of Mammalian Methionine Sulfoxide Reductase B1 Due to Intronization and Protein Processing

Background: Methionine sulfoxide reductases (Msrs) are repair enzymes that protect proteins from oxidative stress by catalyzing stereospecific reduction of oxidized methionine residues. MsrB1 is a selenocysteine-containing cytosolic/nuclear Msr with high expression in liver and kidney. Principal Findings: Here, we identified differences in MsrB1 gene structure among mammals. Human MsrB1 gene consists of four, whereas the corresponding mouse gene of five exons, due to occurrence of an additional intron that flanks the stop signal and covers a large part of the 3′-UTR. This intron evolved in a subset of rodents through intronization of exonic sequences, whereas the human gene structure represents the ancestral form. In mice, both splice forms were detected in liver, kidney, brain and heart with the five-exon form being the major form. We found that both mRNA forms were translated and supported efficient selenocysteine insertion into MsrB1. In addition, MsrB1 occurs in two protein forms that migrate as 14 and 5 kDa proteins. We found that each mRNA splice form generated both protein forms. The abundance of the 5 kDa form was not influenced by protease inhibitors, replacement of selenocysteine in the active site or mutation of amino acids in the cleavage site. However, mutation of cysteines that coordinate a structural zinc decreased the levels of 5 and 14 kDa forms, suggesting importance of protein structure for biosynthesis and/stability of these forms. Conclusions: This study characterized unexpected diversity of protein and mRNA forms of mammalian selenoprotein MsrB1

The host ubiquitin-dependent segregase VCP/p97 is required for the onset of human cytomegalovirus replication

The human cytomegalovirus major immediate early proteins IE1 and IE2 are critical drivers of virus replication and are considered pivotal in determining the balance between productive and latent infection. IE1 and IE2 are derived from the same primary transcript by alternative splicing and regulation of their expression likely involves a complex interplay between cellular and viral factors. Here we show that knockdown of the host ubiquitin-dependent segregase VCP/p97, results in loss of IE2 expression, subsequent suppression of early and late gene expression and, ultimately, failure in virus replication. RNAseq analysis showed increased levels of IE1 splicing, with a corresponding decrease in IE2 splicing following VCP knockdown. Global analysis of viral transcription showed the expression of a subset of viral genes is not reduced despite the loss of IE2 expression, including UL112/113. Furthermore, Immunofluorescence studies demonstrated that VCP strongly colocalised with the viral replication compartments in the nucleus. Finally, we show that NMS-873, a small molecule inhibitor of VCP, is a potent HCMV antiviral with potential as a novel host targeting therapeutic for HCMV infection

Gene and genon concept: coding versus regulation: A conceptual and information-theoretic analysis of genetic storage and expression in the light of modern molecular biology

We analyse here the definition of the gene in order to distinguish, on the basis of modern insight in molecular biology, what the gene is coding for, namely a specific polypeptide, and how its expression is realized and controlled. Before the coding role of the DNA was discovered, a gene was identified with a specific phenotypic trait, from Mendel through Morgan up to Benzer. Subsequently, however, molecular biologists ventured to define a gene at the level of the DNA sequence in terms of coding. As is becoming ever more evident, the relations between information stored at DNA level and functional products are very intricate, and the regulatory aspects are as important and essential as the information coding for products. This approach led, thus, to a conceptual hybrid that confused coding, regulation and functional aspects. In this essay, we develop a definition of the gene that once again starts from the functional aspect. A cellular function can be represented by a polypeptide or an RNA. In the case of the polypeptide, its biochemical identity is determined by the mRNA prior to translation, and that is where we locate the gene. The steps from specific, but possibly separated sequence fragments at DNA level to that final mRNA then can be analysed in terms of regulation. For that purpose, we coin the new term “genon”. In that manner, we can clearly separate product and regulative information while keeping the fundamental relation between coding and function without the need to introduce a conceptual hybrid. In mRNA, the program regulating the expression of a gene is superimposed onto and added to the coding sequence in cis - we call it the genon. The complementary external control of a given mRNA by trans-acting factors is incorporated in its transgenon. A consequence of this definition is that, in eukaryotes, the gene is, in most cases, not yet present at DNA level. Rather, it is assembled by RNA processing, including differential splicing, from various pieces, as steered by the genon. It emerges finally as an uninterrupted nucleic acid sequence at mRNA level just prior to translation, in faithful correspondence with the amino acid sequence to be produced as a polypeptide. After translation, the genon has fulfilled its role and expires. The distinction between the protein coding information as materialised in the final polypeptide and the processing information represented by the genon allows us to set up a new information theoretic scheme. The standard sequence information determined by the genetic code expresses the relation between coding sequence and product. Backward analysis asks from which coding region in the DNA a given polypeptide originates. The (more interesting) forward analysis asks in how many polypeptides of how many different types a given DNA segment is expressed. This concerns the control of the expression process for which we have introduced the genon concept. Thus, the information theoretic analysis can capture the complementary aspects of coding and regulation, of gene and genon

Building bridges between basic science and clinical medicine: a liberal arts perspective

A critical issue for improving global health care is to better integrate basic science and clinical practice, as such integration will lead to innovative solutions. In this article, I will present models for how to prepare students to participate effectively on multidisciplinary teams that foster cooperation between scientists, medical centers, biotechnology businesses, and governmental bodies. I will provide examples of training programs in the United States (USA) designed to increase the number of and diversity of scientists and clinicians engaged in bridging basic science and clinical medicine, also called translational research. The training programs target different stages in career development, from pre-medical students through early career faculty, and have varied organisational structures. Many of the programs have existed long enough for institutions to be able to evaluate their effectiveness, and despite the different program contexts, there are key characteristics common to all of the programs that correlate with successful outcomes. Many of these characteristics can be adapted to other career stages and settings. I will summarize these and describe an example of an interdisciplinary, integrated science course for undergraduates that introduces students at the earliest stage of their careers to addressing complex problems through teamwork. Finally, I will provide suggestions for how other institutions can implement training programs that will build bridges between basic science and clinical medicine