Diamond Blackfan Anemia: An RPS19 Transcript Variant Specifically Interacts with Nuclear Proteins.

Diamond Blackfan Anemia (DBA) is a rare congenic anemia characterized by reduced erythrocyte precursors. Mutations in the ribosomal protein S19 (RPS19) gene are found in about 20% of patients, although it is unclear how this relates to the pathophysiology of the disease. Two transcripts have been reported for human RPS19 in the NCBI database (BC000023 and BC018616), which only differ in the length of the 5′UTR. Both transcripts predict translation to the same protein product and we therefore speculate that the extended 5′UTR may be of importance. While the short (569bp) transcript is well studied, the long (873bp) transcript has not been investigated. We determined the size and tissue distribution as a basic characterization of the long RPS19 transcript. Furthermore, we examined proteins interacting with the different RPS19 transcript variants. Two methods were used determine the size of long RPS19 transcript. Ribonuclease protection assays and 5′-RACE indicate a transcript length consistent with the reported sequence with a 5′UTR of 372bp. Interestingly the 5′UTR predicted in the long transcript contains sequence for a previously reported promoter, suggesting a second novel promoter. Tissue distribution was determined using reverse transcriptase PCR on isolated RNA from a panel of tissues including heart, bone marrow, fetal liver, liver, brain, fetal brain, muscle, kidney, uterus, adrenal gland, lungs, placenta, prostate testes, ovary, thymus, colon, lymphocytes and from K562 and HeLa cell lines. Analysis indicated a ubiquitously expression of the long RPS19 transcript. In order to investigate interactions between the RPS19 transcripts and regulatory proteins, a UV cross-linking assay was performed using in vitro transcribed RNA representing both short and long transcripts. A protein of 55 kDa from nuclear fractions prepared from the erythroid cell lines K562, UT-7 and Cos-1 cells was found to bind to the long RPS19 transcript but not to the short RPS19 transcript. Our results provide evidence for an additional long, RPS19 transcript containing an extended 5′UTR that is ubiquitously transcribed and indicates the existence of a second promoter for RPS19. We further show that a nuclear protein, present in two different erythroid cell lines, interacts with the long RPS19 transcript indicating a function for the extended 5′UTR. This data suggests additional regulatory pathways involving RPS19 mRNA which may be of relevance to the understanding of the molecular mechanisms behind DBA.