Cis-acting short sequence motifs play important roles in alternative splicing. It is now possible to identify such sequence motifs as conserved sequence patterns in genome sequence alignments. Here, we report the systematic search for motifs in the neighboring introns of alternatively spliced exons by using comparative analysis of mammalian genome alignments. We identified 11 conserved sequence motifs that might be involved in the regulation of alternative splicing. These motifs are not only significantly overrepresented near alternatively spliced exons, but they also co-occur with each other, thus, forming a network of cis-elements, likely to be the basis for context-dependent regulation. Based on this finding, we applied the motif co-occurrence to predict alternatively skipped exons. We verified exon skipping in 29 cases out of 118 predictions (25%) by EST and mRNA sequences in the databases. For the predictions not verified by the database sequences, we confirmed exon skipping in 10 additional cases by using both RT-PCR experiments and the publicly available RNA-Seq data. These results indicate that even more alternative splicing events will be found with the progress of large-scale and high-throughput analyses for various tissue samples and developmental stages.

PAL2NAL is a web server that constructs a multiple codon alignment from the corresponding aligned protein sequences. Such codon alignments can be used to evaluate the type and rate of nucleotide substitutions in coding DNA for a wide range of evolutionary analyses, such as the identification of levels of selective constraint acting on genes, or to perform DNA-based phylogenetic studies. The server takes a protein sequence alignment and the corresponding DNA sequences as input. In contrast to other existing applications, this server is able to construct codon alignments even if the input DNA sequence has mismatches with the input protein sequence, or contains untranslated regions and polyA tails. The server can also deal with frame shifts and inframe stop codons in the input models, and is thus suitable for the analysis of pseudogenes. Another distinct feature is that the user can specify a subregion of the input alignment in order to specifically analyze functional domains or exons of interest. The PAL2NAL server is available at http://www.bork.embl.de/pal2nal.

Four years after the original sequence submission, we have re-annotated the genome of Mycoplasma pneumoniae to incorporate novel data. The total number of ORFss has been increased from 677 to 688 (10 new proteins were predicted in intergenic regions, two further were newly identified by mass spectrometry and one protein ORF was dismissed) and the number of RNAs from 39 to 42 genes. For 19 of the now 35 tRNAs and for six other functional RNAs the exact genome positions were re-annotated and two new tRNA(Leu) and a small 200 nt RNA were identified. Sixteen protein reading frames were extended and eight shortened. For each ORF a consistent annotation vocabulary has been introduced. Annotation reasoning, annotation categories and comparisons to other published data on M.pneumoniae functional assignments are given. Experimental evidence includes 2-dimensional gel electrophoresis in combination with mass spectrometry as well as gene expression data from this study. Compared to the original annotation, we increased the number of proteins with predicted functional features from 349 to 458. The increase includes 36 new predictions and 73 protein assignments confirmed by the published literature. Furthermore, there are 23 reductions and 30 additions with respect to the previous annotation. mRNA expression data support transcription of 184 of the functionally unassigned reading frames.