Alex V. Kochetov

Alternative translation start sites and hidden coding potential of eukaryotic mRNAs

It is widely suggested that a eukaryotic mRNA typically contains one translation start site and encodes a single functional protein product. However, according to current points of view on translation initiation mechanisms, eukaryotic ribosomes can recognize several alternative translation start sites and the number of experimentally verified examples of alternative translation is growing rapidly. Also, the frequent occurrence of alternative translation events and their functional significance are supported by the results of computational evaluations. The functional role of alternative translation and its contribution to eukaryotic proteome complexity are discussed.

Eukaryotic mRNAs encoding abundant and scarce proteins are statistically dissimilar in many structural features.

It is well known that non‐coding mRNA sequences are dissimilar in many structural features. For individual mRNAs correlations were found for some of these features and their translational efficiency. However, no systematic statistical analysis was undertaken to relate protein abundance and structural characteristics of mRNA encoding the given protein. We have demonstrated that structural and contextual features of eukaryotic mRNAs encoding high‐ and low‐abundant proteins differ in the 5′ untranslated regions (UTR). Statistically, 5′ UTRs of low‐expression mRNAs are longer, their guanine plus cytosine content is higher, they have a less optimal context of the translation initiation codons of the main open reading frames and contain more frequently upstream AUG than 5′ UTRs of high‐expression mRNAs. Apart from the differences in 5′ UTRs, high‐expression mRNAs contain stronger termination signals. Structural features of low‐ and high‐expression mRNAs are likely to contribute to the yield of their protein products.

The role of alternative translation start sites in the generation of human protein diversity

According to the scanning model, 40S ribosomal subunits initiate translation at the first (5′ proximal) AUG codon they encounter. However, if the first AUG is in a suboptimal context, it may not be recognized, and translation can then initiate at downstream AUG(s). In this way, a single RNA can produce several variant products. Earlier experiments suggested that some of these additional protein variants might be functionally important. We have analysed human mRNAs that have AUG triplets in 5′ untranslated regions and mRNAs in which the annotated translational start codon is located in a suboptimal context. It was found that 3% of human mRNAs have the potential to encode N-terminally extended variants of the annotated proteins and 12% could code for N-truncated variants. The predicted subcellular localizations of these protein variants were compared: 31% of the N-extended proteins and 30% of the N-truncated proteins were predicted to localize to subcellular compartments that differed from those targeted by the annotated protein forms. These results suggest that additional AUGs may frequently be exploited for the synthesis of proteins that possess novel functional properties.

uORFs, reinitiation and alternative translation start sites in human mRNAs

It is known that eukaryotic ribosomes are able to translate small ORFs and reinitiate translation at downstream start codons. However, this mechanism is widely considered to be inefficient and it is not commonly taken into account. We compiled a sample of human mRNAs containing small upstream ORFs overlapping with annotated protein coding sequences. Statistical analysis supported the hypothesis on reinitiation of translation at downstream AUG codons and functional significance of potential alternative ORFs. It may be assumed that some 5′UTR‐located upstream ORFs can deliver ribosomes to alternative translation starts, and they should be taken into consideration in the prediction of human mRNA coding potential.

Protection of transgenic tobacco plants expressing bovine pancreatic ribonuclease against tobacco mosaic virus

Transgenic tobacco plants (Nicotiana tabacum cv. SR1) expressing extracellular pancreatic ribonuclease from Bos taurus and characterized by an increased level of ribonuclease activity in leaf extracts were challenged with tobacco mosaic virus. The transgenic plants exhibited a significantly higher level of protection against the virus infection than the control non-transformed plants. The protection was evidenced by the absence (or significant delay) of the appearance of typical mosaic symptoms and the retarded accumulation of infectious virus and viral antigen. These results demonstrate that modulation of extracellular nuclease expression can be efficiently used in promoting protection against viral diseases.

Interrelations between the nucleotide context of human start AUG codon, N-end amino acids of the encoded protein and initiation of translation.

Abstract It is known that the recognition of AUG triplet by eukaryotic ribosomes as a translation start site strongly depends on its nucleotide context. However, the relative significance of different context positions is not fully clear. In particular, it concerns the role of 3′-end part of the context located at the beginning of the protein-coding sequence. The significant bias observed in nucleotide frequencies in positions +4, +5, +6 (corresponding to the second codon of CDS) could result from different reasons and their contribution to start codon recognition and initiation of translation is under discussion. In this study, we conducted a comparative computational analysis of the human mRNA samples containing different nucleotides (adenine, guanine or pyrimidine) in the essential context position −3. It was found that the presence of G in position +4 could be important for the context variant GnnAUG but not for AnnAUG. Interestingly, the second position of proteins encoded by mRNAs with AnnAUG context variant was specifically and significantly enriched with serine whereas the presence of GnnAUG context also correlated with a higher occurrence of alanine and glycine. It is likely that the efficiency of translation initiation process can depend on the interplay between 5′-context part, 3′-context part and the type of amino acid in the second position of the encoded protein.

Simple database to select promoters for plant transgenesis

The experiments with transgenic plants frequently demand selection of promoters providing appropriate transcription patterns. The set of promoters commonly used in vectors and genetic constructs is very limited, and these promoters provide only a few variants of gene expression patterns. Moreover, identical promoters in a complex construct can induce transgene silencing. This problem can be solved using a variety of plant gene promoters with experimentally verified characteristics. However, this requires a time-consuming analysis of literature data. Here, we describe a database of plant promoters (TransGene Promoters, TGP; http://wwwmgs.bionet.nsc.ru/mgs/dbases/tgp/home.html). TGP contains the information on genomic DNA segments providing certain expression patterns of reporter genes in experiments with transgenic plants. TGP was constructed on the SRS platform, and its interface allows users to search for the promoters with particular characteristics.

Interrelations between the efficiency of translation start sites and other sequence features of yeast mRNAs

The translation start site (TSS) plays an important role in the control of the translational efficiency and cytoplasmic stability of eukaryotic mRNAs. The efficiency of TSS recognition is known to be influenced by sequence context, and mRNAs with “weak” TSSs are relatively abundant. We analyzed a sample of 4113 yeast genes in a search for features that might serve to compensate for the inefficient recognition of “weak” TSSs by initiating ribosomes. The first feature found to correlate with variations in TSS strength is differences in the stability of secondary structure upstream and downstream of the start AUG codon. The second feature concerns the characteristics of AUG triplets found at the beginning of the coding sequence, i.e., downstream of the predicted TSS. In particular, the proximal downstream AUG lies in frame with the CDS significantly more often if the TSS itself is located in a “weak” context. The accuracy of TSS annotation, the possibility of polypeptide heterogeneity due to the use of alternative downstream AUGs, and the influence of related features of mRNA sequences are discussed.

Hidden coding potential of eukaryotic genomes: nonAUG started ORFs

It is widely considered that the vast majority of eukaryotic mRNAs contain only one open reading frame (ORF) and encode single protein. However, eukaryotic ribosomes can initiate translation at alternative start codons due to leaky scanning or reinitiation mechanisms that provides an opportunity to synthesize several protein products. Recent investigations also demonstrated that alternative translation from nonAUG start codons and AUG codons in a weak nucleotide context could make an important contribution to eukaryotic proteomes. However, accurate prediction of alternative start codons demands detailed investigation of mRNA features influencing their recognition by eukaryotic ribosomes. In this work, we present the results of computational analysis of characteristics of yeast and mammalian mRNAs potentially involved in the recognition of nonAUG start codons. It was found that sequence features of nonAUG started Saccharomyces cerevisiae upstream ORFs (uORFs) were adjusted for efficient translation and these uORFs could frequently encode functional polypeptides. In particular, our initial studies revealed that predicted tertiary structures downstream of nonAUG start sites in mammalian mRNAs were energetically more stable than those predicted for AUG start sites with strong Kozak context. We hypothesize that presence of such stable tertiary structure downstream of nonAUG start sites could be an important factor for the ribosome to recognize noncanonical start codons.

On nucleotide solvent accessibility in RNA structure.

Sequence dependence of solvent accessibility in globular and membrane proteins is well established. However, this important structural property has been poorly investigated in nucleic acids. On the other hand investigation of structural determinants of transcriptional and post-transcriptional processes in gene expression are also in a primitive stage and there is a need to explore novel sequence and structural features of both DNA and RNA, which may explain both basic and regulatory mechanisms at various stages of expression. We have recently shown that the nucleotide accessibility in double-stranded DNA molecules strongly depends on sequence context and can be predicted using neighbor information. In this work, we investigate statistics, neighbor-dependence and predictability of nucleotide solvent accessibility for various types of RNA molecules (single-stranded, double-stranded, protein-unbound and protein-bound). It was found that average solvent accessibility of different RNA trinucleotides varies considerably. Interestingly, important translational signals (initiatory AUG codon, Shine-Dalgharno site) were characterized by high solvent accessibility that could be important for its selection in evolution. We also analyzed a relationship between nucleotide accessibility and synonymous codon usage bias in some genomes and find that the two properties are directly related. We believe that the analysis and prediction of nucleotide solvent accessibility opens new avenues to explore more biologically meaningful relationship between RNA structure and function.