Temple logo Dr. Richard Waring

Department of Biology
Temple University


Contact Information
Department of Biology  
Temple University
Philadelphia
PA 19122

Tel: 215-204-8877
Fax: 215-204-6646
e-mail: waring@temple.edu
http://unix.temple.edu/~waring/

Research Interests

Frequently during RNA processing, sections of RNA, called introns, must be spliced out and the flanking sequences rejoined. A type of intron known as Group I is remarkable in that the intron RNA is actually the catalyst for the excision reaction (Cech et al., 1981). RNA catalysts are known as ribozymes.

My laboratory studies group I introns. Some group I introns can perform this reaction in vitro and are said to be self-splicing. Others require the assistance of proteins; the few identified thus far stabilize or help fold the intron RNA (Weeks and Cech, 1996; Caprara et al., 2001). Mutational analysis has shown that the excision of some group I introns depends upon a maturase protein encoded by the intron itself (Lazowska et al., 1980). The first biochemical assay for maturase activity was developed in our laboratory: a maturase from Aspergillus nidulans directly and significantly facilitates excision of an intron with limited self-splicing activity (Ho et al, 1997). The intron (AnCOB) is located in the apocytochrome b gene.  A  high resolution secondary structure may be seen as a pdf file.

Interestingly the maturase protein can also be classified as belonging to a group of proteins known as DNA homing endonucleases (Dujon, 1989). These are generally encoded in introns (or inteins) of about 1,000 base pairs. The proteins recognize (or home in on) a target sequence of 14 - 40 base pairs that corresponds to an intronless version of the DNA sequence flanking their own intron. They cleave this site and initiate the process of inserting a copy of their intron sequence into the cut site. By convention the AnCOB intron-encoded homing endonuclease is named I-AniI.
A large number of DNA endonucleases similar to I-AniI have been identified but the majority do not appear to have maturase activity. The defining characteristics of a maturase are not evident from sequence analysis. It is commonly believed that the RNA splicing activity evolved from a protein which originally functioned solely as a DNA endonuclease (Belfort, 1990; Lambowitz and Perlman, 1990). However it is unlikely that this new function arose simply by fusion of an additional gene sequence. The raises the general question "how does a protein acquire a new function without increasing in size and how easily can this can be detected?". This is particularly relevant to annotating genomes. Attribution of function to a gene on the basis of similarity of either amino acid sequence or tertiary structure to a previously characterized protein may constitute only partial characterization of the sequence since an unknown number of proteins may "moonlight" and perform a second, unrelated and unforeseen task (Jeffery, 1999).

We are curious to learn more about how such proteins develop an additional function. Intriguingly a number of well-known transcription factors not only bind DNA but also have RNA-binding properties (Cassiday and Maher, 2002). It is clearly of interest to understand the origins and the structures of RNA and DNA binding sites on the same protein. Biochemical and genetic data from our lab suggest that the two sites for binding RNA and DNA on I-AniI are distinct although there may be some overlap (Geese et al., 2003; Bolduc et al., 2003).

Dr. Barry Stoddard (Fred Hutchinson Cancer Research Center) along with Dr Mark Caprara (Center for RNA Molecular Biology, Case Western Reserve University Medical School) have collaborated with us to solve the molecular structure of I-AniI complexed with its DNA substrate at a resolution of 2.6 Å (Bolduc et al., 2003). The right figure marks regions of clustered positively charged amino acids conserved between three maturases. These are potential regions on the protein that might be utilized to bind RNA and one has been identified as specifically required for RNA splicing but not DNA cleavage (Bolduc et al., 2003). The overall three dimensional structures of I-AniI and I-CreI, a prototype homing endonuclease (Heath et al., 1997), are surprisingly similar given the dissimilarity of their amino acid sequences. This now makes identifying the RNA binding determinants of I-AniI all the more intriguing since I-CreI does not have maturase activity. We are now particularly interested in determining the molecular structure of the maturase complexed to its group I intron RNA.

Complex of I-AniI and DNA substrate conserved positively charged amino acids















Our system allows both group I maturase RNA splicing and DNA endonuclease activities to be assayed in the same protein in vitro. Furthermore the catalytic activity of the RNA can be assayed without the protein because it retains residual self-splicing activity. The maturase helps fold the intron RNA into its correct three dimensional structure. It recognizes a poorly folded RNA, stimulating the maximum rate of splicing under conditions where tertiary interactions and some long-range helical pairings (secondary structure) are unstable prior to binding of the protein (Ho and Waring, 1999). Deletion analysis indicates that an almost completely intact RNA tertiary structure is required for tight binding, indicating that the protein does not associate predominantly with a subdomain of the RNA (Geese and Waring, 2001). We believe that sub-domains of RNA bind highly cooperatively to the protein and each other, making splicing particularly sensitive to RNA deletions.

Our model is that the maturase recognizes a relatively unfolded form of the RNA and then rapidly promotes cooperative formation of the RNA-protein complex. The folding landscape is likely to be fairly smooth without any major rate-limiting step or kinetic traps. RNA molecules frequently get trapped in the wrong conformation and group I introns are no exception; however AnCOB maturase-assisted folding is generally very efficient with <5% of the RNA remaining unreacted. The protein retains several properties characteristic of homing endonucleases which suggests that its DNA binding domain has not undergone dramatic structural adaptations to function as an RNA binding protein.


Cited references (selective)

Cech,T.R., Zaug,A.J., and Grabowski,P.J. (1981). In vitro splicing of the ribosomal RNA precursor of Tetrahymena: involvement of a guanosine nucleotide in the excision of the intervening sequence. Cell 27, 487-496.

Weeks,K.M. and Cech,T.R. (1996). Assembly of a ribonucleoprotein catalyst by tertiary structure capture. Science 271, 345-348.

Caprara,M.G., Myers,C.A., and Lambowitz,A.M. (2001). Interaction of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) with the group I intron P4-P6 domain. Thermodynamic analysis and the role of metal ions. J Mol Biol 308, 165-190.

Lazowska,J., Jacq,C., and Slonimski,P.P. (1980). Sequence of intron and flanking exons in wild-type and box3 mutants of cytochrome b reveals an interlaced splicing protein coded by an intron. Cell 22, 333-348.

Dujon,B. (1989). Group I introns as mobile genetic elements: facts and mechanistic speculations--a review. Gene 82, 91-114.

Belfort,M. (1990). Phage T4 introns: self-splicing and mobility. Annu. Rev. Genet. 24, 363-385.

Lambowitz,A.M. and Perlman,P.S. (1990). Involvement of aminoacyl-tRNA synthetases and other proteins in group I and group II intron splicing. Trends. Biochem. Sci. 15, 440-444.

Jeffery,C.J. (1999). Moonlighting proteins. Trends Biochem Sci 24, 8-11.

Cassiday,L.A. and Maher,L.J., III (2002). Having it both ways: transcription factors that bind DNA and RNA. Nucleic Acids Res 30, 4118-4126.

Heath,P.J., Stephens,K.M., Monnat,R.J., Jr., and Stoddard,B.L. (1997). The structure of I-CreI, a Group I intron-encoded homing endonuclease. Nature Struct. Biol. 4, 468-476.

Publications


Ho, Y., Kim, S-J., and Waring, R.B. A protein encoded by a group I intron in Aspergillus nidulans directly assists RNA splicing and is a DNA endonuclease. Proc. Natl. Sci. 94:8994-8999, 1997.Hur, M., Geese W. and Waring, R.B. Self-splicing activity of the mitochondrial group I introns from Aspergillus nidulans and related introns from other species. Curr. Genet. 32:399-407, 1997.

Ho, Y. and Waring, R.B. The maturase encoded by a group I intron from Aspergillus nidulans stabilizes RNA tertiary structure and promotes rapid splicing. J. Molecular Biology 292, 987-1001, 1999.

Geese, W.J. and Waring, R.B. A comprehensive characterization of a group IB intron and its encoded maturase reveals that protein-assisted splicing requires an almost intact intron RNA. J. Molecular Biology 308, 609-622, 2001.

Geese, W.J., Kwon, Y.K., Wen, X., and Waring, R.B. In vitro analysis of the relationship between endonuclease and maturase activities in the bi-functional group I intron-encoded protein, I-AniI. Eur. J. Biochem. 270, 1543-1554. 2003.

Bolduc, J.M., Spiegel, P.C., Chatterjee, P., Brady, K.L., Downing, M.E., Caprara, M.G., Waring, R.B. and Stoddard, B.L. Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endonuclease and group I intron splicing factor. Genes & Dev. 17:2875-2888, 2003.