Characterization of the full length mRNA coding for Lucina pectinata HbIII revealed an alternative polyadenylation site

Abstract

Lucina pectinata is a bivalve mollusk that lives in the Southwestern coast of Puerto Rico and houses intracellular symbiotic bacteria. This peculiar organism contains three types of hemoglobin, each characterized by distinct physico-chemical properties. Hemoglobin I (HbI) is a sulfide-reactive protein that reacts with H(2)S to form ferric hemoglobin sulfide. In contrast, hemoglobin II and III are oxygen-reactive proteins that remain oxygenated in the presence of hydrogen sulfide. The partial coding region contained in the cDNA sequences we have cloned confirmed the L. pectinata HbIII amino sequence reported in the NCBI protein database) with a single amino acid difference (Asn72Asp; AsnE12Asp). The characterization of the full length mRNA coding for L. pectinata HbIII revealed an alternative polyadenylation site and an alternate transcription start site. The open reading frame (ORF) of the HbIII cDNA is composed of 459nts containing 153 codons. The initiation codon is preceded by 62 nts of untranslated region (5'UTR), whereas two 3'UTR regions of 640 nt and 455 nt long were identified, revealing the presence of alternative polyadenylation sites. Isoforms of the 3'UTR of HbIII only differed in the length of their sequences. It has been hypothesized that alternative polyadenylation acts through shortening of mRNA to regulate RNA localization, translation and stability. Interestingly, the HbIII mRNA is the only one of all the hemoglobin mRNAs from L. pectinata characterized so far with more than one 3'UTR. Primer extension products suggest two closely located start sites of HbIII mRNA transcription. We suggest that the L. pectinata hemoglobin genes may be under different cellular controls that direct them to exert their particular functions. These hypotheses need to be tested by functional studies and analysis of the regulatory elements of the cognate genes for L. pectinata hemoglobins.

Hemoglobin of the bloody clam Tegillarca granosa (Tg-HbI) is involved in the immune response against bacterial infection

“…L. pectinata Hb showed low homology and a weak phylogenetic relationship to other molluscan Hbs. The Hb of L. pectinata is sulfide-reactive and is thought to play an important role in transporting sulfide to bacterial symbionts [11,20]. This indicated that L. pectinata Hb might have diverged from the common molluscan Hb at an early stage of evolution, acquiring a unique function for a specialized environmental niche.…”

“…Few previous reports have been published regarding Hb tissue distribution in invertebrates. One study, using RTePCR, showed that L. pectinata HbIII mRNA was expressed in the mantle, adductor muscle, foot, pericardial and ctenidia, but not in the digestive glands, gonad and intestine [11].…”

“…2). In Tg-HbI, 27 residues (Q 5 , I 11 ) in Sc-HbI. Between the two types of Sc-HbI, only one residue in S. kagoshimensis HbI (I 119 ) was replaced by (N 119 ) in Scapharca inaequivalvis.…”

“…In bovine, a peptic Hb hydrolysate was fractioned by semi-preparative reversed-phase highperformance liquid chromatography (HPLC) and some fractions showed antibacterial activity against four types of bacteria [10]. A Hb of the deep sea clam Lucina pectinata is a sulfide-reactive protein that reacts with hydrogen sulfide (H 2 S) to form ferric Hb sulfide, allowing the symbiotic bacteria to sustain rapid hexose synthesis and supply the host animal with its entire carbohydrate nutrition [11].…”

Structure and immune expression analysis of hemoglobin genes from the blood clam Tegillarca granosa

“…The pigment is found simply dissolved (not in corpuscles) in the blood and tissues, and it colors the muscle red. Peculiar functions have been reported in the clam Lucina pectinata, where hemoglobin I is a sulfide-reactive protein that reacts with H 2 S to form ferric hemoglobin sulfide (Rivera et al, 2008).…”