title: Activation of a p44 pseudogene in Anaplasma phagocytophila by bacterial RNA splicing: a novel mechanism for post-transcriptional regulation of a multigene family encoding immunodominant major outer membrane proteins.
abstract: Immunodominant 44 kDa major outer membrane proteins of Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) are encoded by the p44 multigene family. One of the paralogues, p44-18 is predominantly expressed by A. phagocytophila in mammalian hosts, but is downregulated in the arthropod vector. The expression of p44-18 was upregulated in A. phagocytophila cultivated in HL-60 cells at 37 degrees C compared with 24 degrees C. However, the molecular mechanism of such gene expression was unclear, as p44-18 has a pseudogene-like structure, i.e. it lacks an AUG start codon and is out of frame with an upstream overlapping paralogue, p44-1. In the present study, we found that an amplicon detected by reverse transciption-polymerase chain reaction (RT-PCR) [808 basepair (bp)] for the p44-1/p44-18 gene locus was smaller than that detected by PCR with the genomic DNA (1652 bp) in the A. phagocytophila-infected HL-60 cells cultured at 37 degrees C. A circularized RNA molecule corresponding to the 844 bp region missing from the locus in the RT-PCR product was detected by inverse RT-PCR, indicating that this is an intron (designated p44-1 intervening sequence, p44-1 IVS). The splicing event of p44-1 IVS was also observed when the p44-1 IVS-carrying plasmid was introduced into Escherichia coli, suggesting that the splicing is sequence-dependent. Structural analysis and in vitro splicing experiments of p44-1 IVS suggested that this is likely to represent a new class of introns in eubacteria. The primer extension analysis showed the presence of a putative sigma(32)-type promoter in region upstream from p44-1. Collectively, the novel RNA splicing and the temperature-dependent transcription may account for the dominant p44-18 expression in mammals.
mesh_terms: D000595:Amino Acid Sequence; D041081:Anaplasma phagocytophilum / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D016873:Ehrlichiosis / Q000382:microbiology / Q000662:veterinary; D006734:Horse Diseases / Q000382:microbiology; D006736:Horses; D006801:Humans; D016056:Immunodominant Epitopes; D008969:Molecular Sequence Data; D005810:Multigene Family; D011544:Pseudogenes; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D017422:Sequence Analysis, DNA
chemical_list: D001425:Bacterial Outer Membrane Proteins; D016056:Immunodominant Epitopes; C450839:p44 protein, Anaplasma phagocytophila