Characterization of an abundant COL9A1 transcript in the cochlea with a novel 3' UTR: Expression studies and detection of miRNA target sequence

Abstract

EST N66408 represents one of several large unique clusters expressed in the Morton human fetal cochlear cDNA library. N66408 is 575 bp in size and initial BLAST analysis of this sequence showed no homology to any known genes or expressed sequence tags (ESTs) from other organs or tissues. Sequence of the original cochlear clone from which N66408 was derived revealed that the corresponding cDNA was about 700 bp in size, including 125 bp at its 5' end with homology to the 3' end of COL9A1 in addition to 575 bp of novel sequence. RT-PCR analysis using primers specific to COL9A1 isoforms 1 and 2 detected expression of both isoforms in human fetal cochlea. Tissue in situ hybridization using the novel 3' UTR sequence as probe showed abundant expression in spiral limbus and spiral ligament, and a moderate level of expression in the organ of Corti. dbEST analysis of ESTs specific to the 3' UTR of COL9A1 showed 19 ESTs derived from various tissues; three polyadenylation sites were identified and the majority of these ESTs were derived from overlapping polyadenylation signals at the second site (position 749-758). Comparison of the 3' UTR of human COL9A1 with its orthologs as well as with dbEST uncovered a highly conserved region around the overlapping polyadenylation signals at position 749-758 in mammals. A search of the microRNA database revealed a highly conserved target sequence for miR-9 immediately preceding the overlapping polyadenylation signals in the novel 3' UTR of COL9A1, suggesting its role in posttranscriptional regulation of COL9A1.

Fig. 1

(a) Schematic representation of COL9A1. I. Long form of COL9A1. Vertical boxes represent coding exons. The 5′ region of the long form encoded by exons 1–7 spliced to exon 8 encodes an amino-terminal nontriple-helical domain of 268 residues, including an NC4 domain of 245 amino acids and a putative signal peptide of 23 amino acids. II. Short form of COL9A1. The 5′ region is encoded by an alternative exon 1* present in intron 6 and noncoding exon 7 of the long form spliced to exon 8; the resulting protein lacks the NC4 domain and contains only 25 amino acid residues of nontriple-helical sequence at the amino terminal end. III. Short form of COL9A1 with novel 3′ UTR expressed in human fetal cochlea. IV. Alignment of EST N66408 to the isoform of COL9A1 detected in human fetal cochlea (III). (b) Nucleotide sequence of partial cDNA of COL9A1. Sequence in bold lower case type represents the novel 3′ UTR discovered in the human fetal cochlear cDNA library. tga (bold and italized) denotes the stop codon. Capitalized and italicized letters indicate the three putative polyadenylation signals.

Fig. 2

(A) Autoradiogram of Northern blot analysis of human fetal RNA samples hybridized with EST N66408. High level expression was detected in cochlea; low level expression was observed in testis and kidney after an extended exposure of the blot (not shown). (B) RT-PCR analysis of novel 3′ UTR of COL9A1 shows expression in all tissues except placenta. (C) RT-PCR analysis of the long form of COL9A1 with isoform-specific primers detects expression in cochlea, trachea, skull, and patella. (D) RT-PCR analysis of the short form of COL9A1 using isoform-specific primers reveals expression in all tissues except placenta. (E) RT-PCR analysis of GAPDH demonstrates bands of equal intensity in all tissues.

Fig. 3

Tissue in situ hybridization of novel 3′ UTR sequence of the short form of COL9A1 on human fetal cochlear sections. (A) and (B) show cross sections (4.5×) of the human cochlear duct at 20 weeks gestational age. Images A and B, captured in bright and dark field, respectively, show abundant COL9A1 expression in the spiral limbus and spiral ligament. (C) Magnified image (20×), in bright field, showing expression of the novel 3′ UTR transcript in the spiral ligament and organ of Corti. (D) Expression of the 3′ UTR of COL9A1 transcripts in the spiral ligament, organ of Corti and spiral limbus captured in dark and bright field (magnified 20×). The red color represents the signals captured in dark field and pseudocolored, and the blue color indicates the tissue morphology, stained with hematoxylin and eosin, captured in bright field and pseudocolored.

Fig. 4

(a) Alignment of ESTs specific to the 3′ UTR of COL9A1. The top line in red represents the query 3′ UTR sequence of COL9A1 obtained from the genomic sequence and the boxes within the red line show the position of the potential polyadenylation signals. The dark blue box represents the AATAAA hexamer, the light blue boxes indicate the positions of hexameric variants, and the green box shows the ATTAAA signal at position 839–844. Overlapping light and dark blue boxes represent the overlapping polyadenylation signals AAGAAATAAA at position 749–758. The vertical broken line denotes the putative polyadenylation sites and indicates the first “A” nucleotide in the poly (A) stretch. (b) Highly conserved 3′ UTR of COL9A1 in mammals. The blue underlined region represents the highly conserved miR-9 target sequence in the novel 3′ UTR and the red underlined region represents the overlapping polyadenylation signals AAGAAATAAA at position 749–758 starting from the first nucleotide immediately following the stop codon. The overlapping polyadenylation signals are not found in the Col9a1-specific EST sequences obtained from cow and pig as AAGCAATAAA is present at this position. Dots denote nucleotide sequence identity, and dashes in the cow and pig sequence represent the end of the available sequence from the corresponding ESTs.