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Down syndrome critical region gene 1-like 2 (DSCR1L2) belongs to the human DSCR1-like gene family, which also includes DSCR1 and DSCR1L1. Both DSCR1 and DSCR1L1 proteins interact with calcineurin, a calcium/calmodulin-dependent phosphatase. To date, no interactor has been described for DSCR1L2. The aim of this work was to perform a first functional study of DSCR1L2 using yeast two-hybrid analysis conducted on a human heart cDNA library. Here, we report the interaction between DSCR1L2 and the human cardiac troponin I (TNNI3), the heart-specific inhibitory subunit of the troponin complex, a central component of the contractile apparatus. This interaction was confirmed by both yeast cotransformation and GST (glutathione-sepharose transferase) fusion protein assay. Moreover, a new DSCR1L2 mRNA isoform, generated by alternative splicing, was identified and cloned in different tissues: it lacks two central exons, encoding the most conserved domains among the DSCR1-like protein family. A quantitative relative reverse transcription-polymerase chain reaction (RT-PCR) assay showed that in heart tissue the normalized expression level ratio for DSCR1L2 and DSCR1L2-E2E5 mRNA isoforms is 3.5 : 1, respectively. The yeast cotransformation and GST fusion protein assay demonstrated the interaction between this new DSCR1L2 variant and the human cardiac troponin I and the prominent role of DSCR1L2 exon 2 in determining binding between both DSCR1L2 isoforms and TNNI3. These data indicate an entirely new role for a DSCR1-like family gene, suggesting a possible involvement of DSCR1L2 in cardiac contraction.

Background CYYR1 is a recently identified gene located on human chromosome 21 whose product has no similarity to any known protein and is of unknown function. Analysis of expressed sequence tags (ESTs) have revealed high human CYYR1 expression in cells belonging to the diffuse neuroendocrine system (DNES). These cells may be the origin of neuroendocrine (NE) tumors. The aim of this study was to conduct an initial analysis of sequence, splicing and expression of the CYYR1 mRNA in human NE tumors. Methods The CYYR1 mRNA coding sequence (CDS) was studied in 32 NE tumors by RT-PCR and sequence analysis. A subtle alternative splicing was identified generating two isoforms of CYYR1 mRNA differing in terms of the absence (CAG- isoform, the first described mRNA for CYYR1 locus) or the presence (CAG+ isoform) of a CAG codon. When present, this specific codon determines the presence of an alanine residue, at the exon 3/exon 4 junction of the CYYR1 mRNA. The two mRNA isoform amounts were determined by quantitative relative RT-PCR in 29 NE tumors, 2 non-neuroendocrine tumors and 10 normal tissues. A bioinformatic analysis was performed to search for the existence of the two CYYR1 isoforms in other species. Results The CYYR1 CDS did not show differences compared to the reference sequence in any of the samples, with the exception of an NE tumor arising in the neck region. Sequence analysis of this tumor identified a change in the CDS 333 position (T instead of C), leading to the amino acid mutation P111S. NE tumor samples showed no significant difference in either CYYR1 CAG- or CAG+ isoform expression compared to control tissues. CYYR1 CAG- isoform was significantly more expressed than CAG+ isoform in NE tumors as well as in control samples investigated. Bioinformatic analysis revealed that only the genomic sequence of Pan troglodytes CYYR1 is consistent with the possible existence of the two described mRNA isoforms. Conclusion A new "subtle" splicing isoform (CAG+) of CYYR1 mRNA, the sequence and the expression of this gene were defined in a large series of NE tumors.

Background All standard methods for cDNA cloning are affected by a potential inability to effectively clone the 5' region of mRNA. The aim of this work was to estimate mRNA open reading frame (ORF) 5' region sequence completeness in the model organism Danio rerio (zebrafish). Results We implemented a novel automated approach (5'_ORF_Extender) that systematically compares available expressed sequence tags (ESTs) with all the zebrafish experimentally determined mRNA sequences, identifies additional sequence stretches at 5' region and scans for the presence of all conditions needed to define a new, extended putative ORF. Our software was able to identify 285 (3.3%) mRNAs with putatively incomplete ORFs at 5' region and, in three example cases selected (selt1a, unc119.2, nppa), the extended coding region at 5' end was cloned by reverse transcription-polymerase chain reaction (RT-PCR). Conclusion The implemented method, which could also be useful for the analysis of other genomes, allowed us to describe the relevance of the "5' end mRNA artifact" problem for genomic annotation and functional genomic experiment design in zebrafish. Open peer review This article was reviewed by Alexey V. Kochetov (nominated by Mikhail Gelfand), Shamil Sunyaev, and Gáspár Jékely. For the full reviews, please go to the Reviewers' Comments section.

The activation of the insulin-like growth factor 1/IGF1 receptor system (IGF1/IGF1R) is a critical event in the transformation and tumorigenicity processes in a wide variety of human tumors. The IGF1/IGF1R system has been recently studied in carcinoid tumors that often arise in the gastrointestinal tract; these tumors are characterized by hypersecretion of bioamines and neuropeptides, leading to functional tumor disease. Two alternatively spliced IGF1R mRNA transcripts have been described to differ by only three nucleotides (CAG) in the coding sequence, resulting in an amino-acid change from the originally described Thr-Gly to an Arg in the extracellular portion of the receptor beta subunit. In transfected Chinese hamster ovary cells, the form without CAG (CAG-) exhibited an approximate 2-fold increase in IGF1 stimulation of activities required for its mitogenic properties. In this study, we examine the relative expression of the two IGF1R mRNA isoforms by a semiquantitative RT-PCR approach using highly standardized conditions, beta-2 microglobulin (B2M) as a reference gene and gel imaging analysis. We analyzed a large series of human neuroendocrine tumors (32 samples) and 9 normal tissues. A significant higher expression of both isoforms in the tumor samples (approximately 2-fold increase) was found, while a constant CAG+/CAG- IGF1R mRNA isoforms of an approximate 3:1 ratio was observed in all tumoral and normal cell types studied. The phylogenetic study of the IGF1R locus in several species suggests that human IGF1R CAG- mRNA isoform is evolutionarily more recent compared to the IGF1R CAG+ mRNA isoform and it could be used by the splicing apparatus at this intron/exon junction with a lower efficiency. This study highlights the relevance of IGF1R mRNA expression in neuroendocrine tumor cells, and the constant presence of 'subtle' alternative splicing for the IGF1R locus.

The amino acid sequence of gene products is routinely deduced from the nucleotide sequence of the relative cloned cDNA, according to the rules for recognition of start codon (first-AUG rule, optimal sequence context) and the genetic code. From this prediction stem most subsequent types of product analysis, although all standard methods for cDNA cloning are affected by a potential inability to effectively clone the 5′ region of mRNA. Revision by bioinformatics and cloning methods of 109 known genes located on human chromosome 21 (HC 21) shows that 60 mRNAs lack any in-frame stop upstream of the first-AUG, and that in five cases (DSCR1, KIAA0184, KIAA0539, SON, and TFF3) the coding region at the 5′ end was incompletely characterized in the original descriptions. We describe the respective consequences for genomic annotation, domain and ortholog identification, and functional experiments design. We have also analyzed the sequences of 13,124 human mRNAs (RefSeq databank), discovering that in 6448 cases (49%), an in-frame stop codon is present upstream of the initiation codon, while in the other 6676 mRNAs (51%), identification of additional bases at the mRNA 5′ region could well reveal some new upstream in-frame AUG codons in the optimal context. Proportionally to the HC 21 data, about 550 known human genes might thus be affected by this 5′ end mRNA artifact. © 2003 Elsevier B.V. All rights reserved.