Evolutionary relationships among group II intron-encoded proteins and identification of a conserved domain that may be related to maturase function

Nucleic Acids Research, 1993, Vol. 21, No. 22 4991-4997 Evolutionary relationships among group II intron-encoded proteins and identification of a conserved domain that may be related to maturase function Georg Mohr, Philip S.Perlman1 and Alan M.Lambowitz* Departments of Molecular Genetics, Biochemistry, and Medical Biochemistry, and the Biotechnology Center, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210 and 1 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75235-9038, USA Received July 16, 1993; Revised and Accepted August 23, 1993 Many group II introns encode reverse transcriptase-like proteins that potentially function in intron mobility and RNA splicing. We compared 34 intron-encoded open reading frames and four related open reading frames that are not encoded in introns. Many of these open reading frames have a reverse transcriptase-like domain, followed by an additional conserved domain X, and a Zn2+-finger-like region. Some open reading frames have lost conserved sequence blocks or key amino acids characteristic of functional reverse transcriptases, and some lack the Zn2+-finger-like region. The open reading frames encoded by the chloroplast tRNA1-*8 genes and the related Epifagus virginiana matK open reading frame lack a Zn2+-finger-like region and have only remnants of a reverse transcriptase-like domain, but retain a readily identifiable domain X. Several findings lead us to speculate that domain X may function in binding of the intron RNA during reverse transcription and RNA splicing. Overall, our findings are consistent with the hypothesis that all of the known group II intron open reading frames evolved from an ancestral open reading frame, which contained reverse transcriptase, X, and Zn 2+ -finger-like domains, and that the reverse transcriptase and Zn2+-finger-like domains were lost in some cases. The retention of domain X in most proteins may reflect an essential function in RNA splicing, which is independent of the reverse transcriptase activity of these proteins. INTRODUCTION Group II introns are of interest both because of their RNAcatalyzed splicing mechanism, which resembles that of nuclear pre-mRNA introns, and because they behave as mobile elements (1,2). Group II introns have been found in fungal and plant mitochondria and in chloroplasts (1), and recently, in the proteobacterium Azotobacter vinelandii and the cyanobacterium Calothrix, which are related to the probable ancestors of mitochondria and chloroplasts, respectively (3). All group II introns have a conserved secondary structure, which consists of six double helical domains radiating from a central wheel, with the two different structural classes, HA and UB, distinguished by specific features (1). The conserved RNA structure catalyzes splicing via formation of a lariat intermediate similar to that formed during the splicing of nuclear pre-mRNA introns (4-6). Although some group II introns self-splice in vitro, they require proteins for efficient splicing in vivo, presumably to help fold the intron RNA into the catalytically-active structure. Some of these proteins are encoded by chromosomal genes, whereas others, 'maturases', are encoded by the introns themselves (7). The mobility of group II introns has been inferred from their location and distribution in different genes (8) and from the existence of 'twintrons' in which one group II intron has integrated into either another group II intron or into a degenerate type of group II intron, referred to as a group HI intron (9,10). The first direct evidence for group II intron mobility came from studies showing that two Saccharomyces cerevisiae (yeast) group II introns {coxl intron 1 and coxl intron 2) insert efficiently during crosses into coxl alleles lacking these introns (11,12). Similar findings have also been reported for Kluyveromyces lactis coxl intron 1, a cognate of yeast coxl intron 2 (13). All three of these mobile group II introns contain a long open reading frame (ORF), which encodes a reverse transcriptase (RT)-like protein. The proteins encoded by the yeast coxl introns 1 and 2 have been shown to be bifunctional: they have an RT activity that may play a role in intron mobility (14), and they also function as maturases in splicing the intron in which they are encoded (15-17). In addition to yeast coxl introns 1 and 2, a number of other group II introns contain ORFs that may encode proteins that function in RNA splicing or intron mobility (8). Some of these ORFs are located in the loop of intron domain IV, whereas others are in-frame with the upstream exon with the bulk of the ORF in the loop of domain IV (1). The yeast coxl intron 2 protein, * To whom correspondence should be addressed at: Department of Molecular Genetics, The Ohio State University, 484 W. Twelfth Avenue, Columbus, OH 43210, USA ABSTRACT 4992 Nucleic Acids Research, 1993, Vol. 21, No. 22 Pro F P 1li'H WA HIV-1 Pol F P T • RNase H C Int • \ 1 i Zn S.c. coxl 12 M-Pcoxl 12 I I f I IV V VI VII N.t. trnK II Figure 1. Comparison of protein domains in the HIV-1 pol gene and ORFs of group n introns S.cerevisiae (yeast) coxl intron 2, M. polymorpha coxl intron 2 and N. tabacum trnK intron 1. Protein domains are indicated by marked areas. [Pro, protease; (Pro), possible protease domain of intron ORFs; Z, domain Z; Pol and RT, reverse transcriptase domain; X, domain X; Zn, Zn2+-finger-like region, Int, integrase]. Conserved sequence blocks I to VII characteristic of RTs are indicated below the RT domains (18,20); parentheses indicate weak, but recognizable matches for the RT sequence blocks (see Fig. 2). Demarcated regions of the HIV-1 protein indicate structural domains (P, palm; F, fingers; T, thumb; C, connection) identified in the X-ray crystallographic structure of the protein (32). Vertical arrows indicate positions of yeast coxl intron 2 mutations, which are associated with the maturase defect in mutant C1082 (Ser648 and Asp675; refs. 14,17). Citations for sequence data are given in Table 1. shown schematically in Fig. 1, as well as most other group II intron ORFs, have a readily identifiable RT domain. This domain generally includes matches for the seven conserved sequence blocks characteristic of RTs (denoted I to VII; refs. 18-20), although several of the ORFs lack some of the conserved regions or key amino acids characteristic of functional RTs (see below). Phylogenetic comparisons indicate that the RT domains of group II intron-encoded proteins belong to a class characteristic of the LINE 1-like or non-long-terminal repeat (non-LTR) family of retroelements. Within this class, the group II intron-encoded proteins comprise a separate subgroup, which is most closely related to the RTs of the Neurospora Mauriceville mitochondrial plasmid and bacterial retrons (19—21). In addition to the RT-like domain, the group II intron ORFs also contain a conserved, upstream domain, Z, (...truncated)