Competing RNA secondary structures are required for mutually exclusive splicing of the Dscam exon 6 cluster

Alternative splicing of eukaryotic pre-mRNAs is an important mechanism for generating proteome diversity and regulating gene expression. The Drosophila melanogaster Down Syndrome Cell Adhesion Molecule (Dscam) gene is an extreme example of mutually exclusive splicing. Dscam contains 95 alternatively spliced exons that potentially encode 38,016 distinct mRNA and protein isoforms. We previously identified two sets of conserved sequence elements, the docking site and selector sequences in the Dscam exon 6 cluster, which contains 48 mutually exclusive exons. These elements were proposed to engage in competing RNA secondary structures required for mutually exclusive splicing, though this model has not yet been experimentally tested. Here we describe a new system that allowed us to demonstrate that the docking site and selector sequences are indeed required for exon 6 mutually exclusive splicing and that the strength of these RNA structures determines the frequency of exon 6 inclusion. We also show that the function of the docking site has been conserved for ~500 million years of evolution. This work demonstrates that conserved intronic sequences play a functional role in mutually exclusive splicing of the Dscam exon 6 cluster.     

Multiple Distinct Splicing Enhancers in the Protein-Coding Sequences of a Constitutively Spliced Pre-mRNA

We have identified multiple distinct splicing enhancer elements within protein-coding sequences of the constitutively spliced human β-globin pre-mRNA. Each of these highly conserved sequences is sufficient to activate the splicing of a heterologous enhancer-dependent pre-mRNA. One of these enhancers is activated by and binds to the SR protein SC35, whereas at least two others are activated by the SR protein SF2/ASF. A single base mutation within another enhancer element inactivates the enhancer but does not change the encoded amino acid. Thus, overlapping protein coding and RNA recognition elements may be coselected during evolution. These studies provide the first direct evidence that SR protein-specific splicing enhancers are located within the coding regions of constitutively spliced pre-mRNAs. We propose that these enhancers function as multisite splicing enhancers to specify 3′ splice-site selection.     

Local absence of secondary structure permits translation of mRNAs that lack ribosome-binding sites

The initiation of translation is a fundamental and highly regulated process in gene expression. Translation initiation in prokaryotic systems usually requires interaction between the ribosome and an mRNA sequence upstream of the initiation codon, the so-called ribosome-binding site (Shine-Dalgarno sequence). However, a large number of genes do not possess Shine-Dalgarno sequences, and it is unknown how start codon recognition occurs in these mRNAs. We have performed genome-wide searches in various groups of prokaryotes in order to identify sequence elements and/or RNA secondary structural motifs that could mediate translation initiation in mRNAs lacking Shine-Dalgarno sequences. We find that mRNAs without a Shine-Dalgarno sequence are generally less structured in their translation initiation region and show a minimum of mRNA folding at the start codon. Using reporter gene constructs in bacteria, we also provide experimental support for local RNA unfoldedness determining start codon recognition in Shine-Dalgarno--independent translation. Consistent with this, we show that AUG start codons reside in single-stranded regions, whereas internal AUG codons are usually in structured regions of the mRNA. Taken together, our bioinformatics analyses and experimental data suggest that local absence of RNA secondary structure is necessary and sufficient to initiate Shine-Dalgarno--independent translation. Thus, our results provide a plausible mechanism for how the correct translation initiation site is recognized in the absence of a ribosome-binding site.     

Thioredoxin 1 as a serum marker for breast cancer and its use in combination with CEA or CA15-3 for improving the sensitivity of breast cancer diagnoses

Background

Influenza B and C are single-stranded RNA viruses that cause yearly epidemics and infections. Knowledge of RNA secondary structure generated by influenza B and C will be helpful in further understanding the role of RNA structure in the progression of influenza infection.

Findings

All available protein-coding sequences for influenza B and C were analyzed for regions with high potential for functional RNA secondary structure. On the basis of conserved RNA secondary structure with predicted high thermodynamic stability, putative structures were identified that contain splice sites in segment 8 of influenza B and segments 6 and 7 of influenza C. The sequence in segment 6 also contains three unused AUG start codon sites that are sequestered within a hairpin structure.

Conclusions

When added to previous studies on influenza A, the results suggest that influenza splicing may share common structural strategies for regulation of splicing. In particular, influenza 3′ splice sites are predicted to form secondary structures that can switch conformation to regulate splicing. Thus, these RNA structures present attractive targets for therapeutics aimed at targeting one or the other conformation.     

Evolution of monoblepharidalean fungi based on complete mitochondrial genome sequences

We have determined the complete mitochondrial DNA (mtDNA) sequences of three chytridiomycete fungi, Monoblepharella15, Harpochytrium94 and Harpochytrium105. Our phylogenetic analysis based on concatenated mitochondrial protein sequences confirms the placement of Mono blepharella15 together with Harpochytrium spp. and Hyaloraphidium curvatum within the taxonomic order Monoblepharidales, with overwhelming support. These four mtDNA sequences encode the standard fungal mitochondrial gene complement and, like certain other chytridiomycete fungi, encode a reduced complement of 7–9 tRNAs, some of which require 5′-tRNA editing to be functional. Highly conserved sequence elements were identified upstream of almost all protein-coding genes in the mtDNAs of Monoblepharella15 and both Harpochytrium species. Finally, a guanosine residue is conserved upstream of the predicted ATG or GTG start codons of almost every protein-coding gene in these genomes. The appearance of this G residue correlates with the presence of a non-canonical cytosine residue at position 37 in the anticodon loop of the mitochondrial initiator tRNAs. Based on the unorthodox features in these four genomes, we propose that a 4 bp interaction between the CAUC anticodon of these tRNAs and GAUG/GGUG codons is involved in translation initiation in monoblepharidalean mitochondria. Intriguingly, a similar interaction may also be involved in mitochondrial translation initiation in the sea anemone Metridium senile.     

Chicken transferrin receptor gene: conservation 3'' noncoding sequences and expression in erythroid cells.

Recombinant clones of the chicken transferrin receptor gene and cDNA have been isolated and sequenced. Two highly conserved regions have been identified in the 3' noncoding sequence of the human and chicken TR gene. The conserved regions include sequences that have been shown to be involved in the iron-dependent regulation of human TR mRNA stability. These sequences can be modeled as two different types of RNA secondary structures, one containing stem-loop structures that are similar to the iron-responsive elements found in ferritin mRNA and the other being a stable, duplex/stem-loop structure. Both forms show considerable similarity between chicken and human mRNA. The expression of TR is developmentally regulated during erythroid maturation, and immature erythroid cells express exceptionally high levels of TR mRNA.     

Recognition of unknown conserved alternatively spliced exons

The split structure of most mammalian protein-coding genes allows for the potential to produce multiple different mRNA and protein isoforms from a single gene locus through the process of alternative splicing (AS). We propose a computational approach called UNCOVER based on a pair hidden Markov model to discover conserved coding exonic sequences subject to AS that have so far gone undetected. Applying UNCOVER to orthologous introns of known human and mouse genes predicts skipped exons or retained introns present in both species, while discriminating them from conserved noncoding sequences. The accuracy of the model is evaluated on a curated set of genes with known conserved AS events. The prediction of skipped exons in the approximately 1% of the human genome represented by the ENCODE regions leads to more than 50 new exon candidates. Five novel predicted AS exons were validated by RT-PCR and sequencing analysis of 15 introns with strong UNCOVER predictions and lacking EST evidence. These results imply that a considerable number of conserved exonic sequences and associated isoforms are still completely missing from the current annotation of known genes. UNCOVER also identifies a small number of candidates for conserved intron retention.     

AGenDA: homology-based gene prediction

We present a www server for homology-based gene prediction. The user enters a pair of evolutionary related genomic sequences, for example from human and mouse. Our software system uses CHAOS and DIALIGN to calculate an alignment of the input sequences and then searches for conserved splicing signals and start/stop codons around regions of local sequence similarity. This way, candidate exons are identified that are used, in turn, to calculate optimal gene models. The server returns the constructed gene model by email, together with a graphical representation of the underlying genomic alignment.     

Conserved secondary structures in Aspergillus

Background

Recent evidence suggests that the number and variety of functional RNAs (ncRNAs as well as cis-acting RNA elements within mRNAs ) is much higher than previously thought; thus, the ability to computationally predict and analyze RNAs has taken on new importance. We have computationally studied the secondary structures in an alignment of six Aspergillus genomes. Little is known about the RNAs present in this set of fungi, and this diverse set of genomes has an optimal level of sequence conservation for observing the correlated evolution of base-pairs seen in RNAs.

Methodology/Principal Findings

We report the results of a whole-genome search for evolutionarily conserved secondary structures, as well as the results of clustering these predicted secondary structures by structural similarity. We find a total of 7450 predicted secondary structures, including a new predicted ∼60 bp long hairpin motif found primarily inside introns. We find no evidence for microRNAs. Different types of genomic regions are over-represented in different classes of predicted secondary structures. Exons contain the longest motifs (primarily long, branched hairpins), 5′ UTRs primarily contain groupings of short hairpins located near the start codon, and 3′ UTRs contain very little secondary structure compared to other regions. There is a large concentration of short hairpins just inside the boundaries of exons. The density of predicted intronic RNAs increases with the length of introns, and the density of predicted secondary structures within mRNA coding regions increases with the number of introns in a gene.

Conclusions/Sigificance

There are many conserved, high-confidence RNAs of unknown function in these Aspergillus genomes, as well as interesting spatial distributions of predicted secondary structures. This study increases our knowledge of secondary structure in these aspergillus organisms.     

Pre-mRNA secondary structure and the regulation of splicing

Nuclear pre-mRNAs must be precisely processed to give rise to mature cytoplasmic mRNAs. This maturation process, known as splicing, involves excision of intron sequences and ligation of the exon sequences. One of the major problems in understanding this process is how splice sites, the sequences which form the boundaries between introns and exons, can be accurately selected. A number of studies have defined conserved sequences within introns which were later shown to interact with small nuclear ribonucleoproteins (snRNPs). However, due to the simplicity of these conserved sequences it has become clear that other elements must be involved and a number of studies have indicated the importance of secondary structures within pre-mRNAs. Using various examples, we shall show that such structures can help to specify splice sites by modifying physical distances within introns or by being involved in the definition of exons and, lastly, that they can be part of the regulation of alternative splicing.     

Conservation of an open-reading frame as an element affecting 5' splice site selection

Splice site selection is a key element of pre-mRNA splicing and involves specific recognition of consensus sequences at the 5(') and 3(') splice sites. Evidently, the compliance of a given sequence with the consensus 5(') splice site sequence is not sufficient to define it as a functional 5(') splice site, because not all sequences that conform with the consensus are used for splicing. We have previously hypothesized that the necessity to avoid the inclusion of premature termination codons within mature mRNAs may serve as a criterion that differentiates normal 5(') splice sites from unused (latent) ones. We further provided experimental support to this idea, by analyzing the splicing of pre-mRNAs in which in-frame stop codons upstream of a latent 5(') splice site were mutated, and showing that splicing using the latent site is indeed activated by such mutations. Here we evaluate this hypothesis by a computerized survey for latent 5(') splice sites in 446 protein-coding human genes. This data set contains 2311 introns, in which we found 10490 latent 5(') splice sites. The utilization of 10045 (95.8%) of these sites for splicing would have led to the inclusion of an in-frame stop codon within the resultant mRNA. The validity of this finding is confirmed here by statistical analyses. This finding, together with our previous experimental results, invokes a nuclear scanning mechanism, as part of the splicing machine, which identifies in-frame stop codons within the pre-mRNA and prevents splicing that could lead to the formation of a prematurely terminated protein.     

Pre-mRNA secondary structures influence exon recognition

The secondary structure of a pre-mRNA influences a number of processing steps including alternative splicing. Since most splicing regulatory proteins bind to single-stranded RNA, the sequestration of RNA into double strands could prevent their binding. Here, we analyzed the secondary structure context of experimentally determined splicing enhancer and silencer motifs in their natural pre-mRNA context. We found that these splicing motifs are significantly more single-stranded than controls. These findings were validated by transfection experiments, where the effect of enhancer or silencer motifs on exon skipping was much more pronounced in single-stranded conformation. We also found that the structural context of predicted splicing motifs is under selection, suggesting a general importance of secondary structures on splicing and adding another level of evolutionary constraints on pre-mRNAs. Our results explain the action of mutations that affect splicing and indicate that the structural context of splicing motifs is part of the mRNA splicing code.     

Cost minimization of ribosomal frameshifts

Properties of mRNA leading regions that modulate protein synthesis are little known (besides effects of their secondary structure). Here I explore how coding properties of leading regions may account for their disparate efficiencies. Trinucleotides that form off frame stop codons decrease costs of ribosomal slippages during protein synthesis: protein activity (as a proxy of gene expression, and as measured in experiments using artificial variants of 5' leading sequences of beta galactosidase in Escherichia coli) increases proportionally to the number of stop motifs in any frame in the 5' leading region. This suggests that stop codons in the 5' leading region, upstream of the recognized coding sequence, terminate eventual translations that sometimes start before ribosomes reach the mRNA's recognized start codon, increasing efficiency. This hypothesis is confirmed by further analyses: mRNAs with 5' leading regions containing in the same frame a start preceding a stop codon (in any frame) produce less enzymatic activity than those with the stop preceding the start. Hence coding properties, in addition to other properties, such as the secondary structure of the 5' leading region, regulate translation. This experimentally (a) confirms that within coding regions, off frame stops increase protein synthesis efficiency by early stopping frameshifted translation; (b) suggests that this occurs for all frames also in 5' leading regions and that (c) several alternative start codons that function at different probabilities should routinely be considered for all genes in the region of the recognized initiation codon. An unknown number of short peptides might be translated from coding and non-coding regions of RNAs.