Nitrilase and Fhit homologs are encoded as fusion proteins in Drosophila melanogaster and Caenorhabditis elegans

Abstract

The tumor suppressor gene FHIT encompasses the common human chromosomal fragile site at 3p14.2 and numerous cancer cell biallelic deletions. To study Fhit function we cloned and characterized FHIT genes from Drosophila melanogaster and Caenorhabditis elegans. Both genes code for fusion proteins in which the Fhit domain is fused with a novel domain showing homology to bacterial and plant nitrilases; the D. melanogaster fusion protein exhibited diadenosine triphosphate (ApppA) hydrolase activity expected of an authentic Fhit homolog. In human and mouse, the nitrilase homologs and Fhit are encoded by two different genes: FHIT and NIT1, localized on chromosomes 3 and 1 in human, and 14 and 1 in mouse, respectively. We cloned and characterized human and murine NIT1 genes and determined their exon-intron structure, patterns of expression, and alternative processing of their mRNAs. The tissue specificity of expression of murine Fhit and Nit1 genes was nearly identical. Because fusion proteins with dual or triple enzymatic activities have been found to carry out specific steps in a given biochemical or biosynthetic pathway, we postulate that Fhit and Nit1 likewise collaborate in a biochemical or cellular pathway in mammalian cells.

Figure 1

Sequence comparison of human, murine, D. melanogaster, and C. elegans Nit1 and Fhit proteins. Identities are shown in black boxes, similarities are shown in shaded boxes. For human and mouse FHIT GenBank accession nos. are U46922 and AF047699, respectively.

Figure 2

Expression of Nit1 and Fhit mRNAs in murine and human tissues. (A) Mouse multiple tissues Northern blot. Lanes 1–8: heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis. (Top) Fhit probe; (Middle) Nit1 probe; (Bottom) actin probe. (B) Human blot, NIT1 probe. Lanes 1–8: heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. (C) Lanes 1 and 2: D. melanogaster adult, D. melanogaster embryo; D. melanogaster NitFhit probe. Lane 3: C. elegans adult; C. elegans NitFhit probe.

Figure 3

Genomic organization of human and murine Nit1 genes and D. melanogaster and C. elegans NitFhit genes. (A) Exon-intron structure of the genes. (B) Alternative processing of human NIT1 gene.

Figure 4

Cleavage of ApppA by D. melanogaster NitFhit. At indicated times of incubation, samples were spotted on TLC plates with appropriate nucleotide standards.

Figure 5

Analysis of alternative transcripts of human NIT1 by RT-PCR. RT-PCR of HeLa RNA was performed with primers in different exons. Lanes 1–6: exons 1 and 3 (transcript 2); exons 1C and 3 (transcript 5); exons 1A and 3 (transcripts 3, upper band and 4, lower band); exons 2 and 3 (transcripts 2–4); exons 1 and 1C (transcript 5); and exons 1 and 2 (transcript 2).