After the challenge supposed by the human genome sequencing, as well as of other organisms with medical or commercial interest, the determination of the transcriptome of these species is a prerequisite for fully understanding not only their molecular biology, but also for translating this information to technical applications in medicine, pharmacology and biotechnology. In this sense, high throughput technologies supported by systematic cDNA libraries sequencing has been the main approach used for transcriptome characterisation. Thus, sequencing of random transcript cDNA clones that results in short partial sequences, known as expressed sequence tags  (EST) or, more recently, methods for full length isolation and sequencing of random clones from cDNA libraries have been used [33, 34]. In addition, in the last few years massive sequencing technologies have also been developed for transcriptome analysis [7, 24, 35]. All these high throughput techniques present the advantage to generate a considerable volume of information in a relative short time, but these techniques seem to be inefficient for discovering relative rare transcripts. This affirmation is supported by recent data that suggest the existence of a wealth of transcripts which had, so far, escaped detection through systematic sequencing of cDNA libraries . In a recent published work  combining EST and RNAseq data, it has been showed that chimeras are lowly expressed transcripts. Thus, here we present that nested RT-PCR shows to be an efficient tool to discover a number of transcripts expressed from a concrete locus, not only those highly expressed but also those expressed at lower levels. In fact, 62 of 76 transcripts detected in our experiments had never been described before. This supposes 81.5% of novel sequences, showing how powerful this technique is in order to detect transcripts from a concrete locus. In this respect, our results show that current gene and transcript annotation sets might cover only a small fraction of the total transcriptional output of the human and other organism described genomes, and in this sense, a major enforce should be taken in order to detect more transcripts of a particular organism.
A detailed human and mouse Ly6g5b transcript analysis has been previously described by our group [13, 16]. However, here we have extended this comparative analysis among different mammalian species, showing that Ly6g5b first intron retention is a ubiquitous and important characteristic conserved in mammals. Its apparent capacity to escape NMD together with its conservation in the mammalian group studied points to an important role for this non-coding RNA, which should be investigated. So, we could conclude that Ly6g5b gene presents a double expression pattern. The first one, quite similar among tissues and species, is constituted by Ly6g5b transcripts with first intron retention event. The second one seems to be tissue and species specific and is constituted by canonical Ly6g5b transcripts with a complete Ly-6 ORF as well as aberrant and non conserved transcripts, with no common pattern distribution.
For Csnk2b gene expression we can also describe a double expression pattern. The first one constituted by the canonical isoform, which is quite similar among tissues and species, and the second one which seems to be tissue and species specific and is constituted by aberrant and non conserved transcripts, with no common pattern distribution.
CSNK2B-LY6G5B human chimerism was initially described by Calvanesse et al (2008) by using different human cell lines . However, here we show the first comparative analysis of this TIC event using RNA from different mammalian tissues and species. Our results show that, far to be a human characteristic, Csnk2b-Ly6g5b chimerism is widely conserved in mammals. Its conservation among all the species analysed in this study shows how Csnk2b-Ly6g5b chimerism is not a trivial event. The majority of them lack the last exon of the upstream gene as well as the first exon of the downstream gene which is consistent with previous reported data [22, 23]. This eliminates the stop codon as well as the molecular targets present in the 3’ UTR region of the transcripts of the upstream gene. We also agree with these authors that run-off is the most likely mechanism involved in the origin of TIC, since some chimeric transcripts detected in our study maintain the intergenic region. Other mechanisms proposed for generating chimeric transcripts, like trans-splicing, are not likely to maintain these intergenic regions.
In addition to the canonical Csnk2b, Ly6g5 and Csnk2b-Ly6g5b transcripts with a coherent ORF, other transcripts detected in our study present exon skipping as well as intron retention events which allow to generate, assuming that all these transcripts could be translated into protein by using canonical start codon, truncated or aberrant proteins. Others are non-coding RNAs. The observation that there are tens of thousands of non-coding RNA (ncRNA) expressed in mammals, and that most of the genome is transcribed, confronts and contradicts the traditional protein-centric view of genetic information and genome organisation , . Thus, there are two opposing alternatives either the bulk of the transcription which does not yield mRNAs is ‘transcriptional noise’ and/or the residue of evolutionary baggage retained or accumulated within genes, or this transcription comprises another level of expression and transaction of RNA information that is important to the evolution and developmental ontogeny of the higher organisms . If one assumes that all this is transcriptional noise and that all these transcripts are the result of transcriptional machinery mistakes while it is working, they should not be distributed in a specific manner.
Some authors defend that chimerism might generate bi-functional proteins having properties from both original proteins . Through different analyses of our results, we could identify chimeras which maintain the ORFs of Csnk2b and Ly6g5b susceptible to form bi-functional chimeric proteins in Homo sapiens, Macaca mulata, and Sus scrofa. The fact that these were not found in cow, rat and mouse does not indicate functional chimera absence in these species, since they could be present in other tissues not analysed in this study. These hypothetical new chimeric proteins all carry N-terminus domains from Csnk2b involved in structural aspects that are required for Csnk2b exportation to the cellular surface  and/or its regulation , as well as Ly-6 domain amino acid sequence [13, 14] at their C-terminus. However, the chimera “bi-functional” protein will affect the juxta-dimer interface region  containing the zinc-finger involved in the homo-dimerisation, as well as all the C-terminal domain involved in the interaction with the CSNK2A subunits and the crucial last 20 amino-acids also involved in the homo-dimerisation. In addition, the Ly-6 domain, with 10 Cysteins, could not be folded as such due to the intracellular localisation. These two facts indicate that the “bi-functional” chimera would not be formed, and would only have one function: the one of CSNK2B, although possibly binding to other kinase different to CSNK2A due to the alterations produced on the C-terminal domain commented above. Other chimeras would only be affected from exon 7, containing then the juxta-dimer interface region but differing at the very end C-terminal region and not containing the Ly-6 domain sequence due to frameshifts, and probably only affecting the binding to CSNK2A. It has been proposed that CSNK2B might bind other kinases such as Ras-1 and Mos to modify their catalytic affinity in a CK2-independent fashion. The alternative C-terminal ends, generated by the chimeric transcripts, could increment the binding repertoire of CSNK2B to other kinases or non-kinase proteins converting CSNK2B in even a wider “wild-card” regulator subunit than previously proposed .
In addition, we have found chimeric transcripts that would encode a complete CSNK2B protein, but with different 3´UTR due to the Ly6g5b sequence, in Macaca mulata, Bos taurus and Rattus norvegicus. These transcripts could have different mRNA localisations or stabilities which could have altered protein functional implications [40, 41]. It has been described that some 3´UTR contain “localization elements” or “zip codes” which target mRNAs to specific subcellular sites.