Justin M Waldern1,2, Dorie Smith1, Carol Lyn Piazza1, E Jake Bailey1, Nicholas J Schiraldi3, Reza Nemati4,5, Dan Fabris1,4,6, Marlene Belfort7,8, Olga Novikova9,10. 1. Department of Biological Sciences and RNA Institute, University at Albany, 1400 Washington Avenue, Albany, NY, 12222, USA. 2. Current address: Department of Biology, University of North Carolina, 270 Bell Tower Drive, Chapel Hill, NC, 27599, USA. 3. Academic and Research Computing Center, Information Technology Services, University at Albany, 1400 Washington Avenue, Albany, NY, 12222, USA. 4. Department of Chemistry, University at Albany, 1400 Washington Avenue, Albany, NY, 12222, USA. 5. Current address: Biogen, 125 Broadway, Cambridge, MA, 02142, USA. 6. Current address: Department of Chemistry, University of Connecticut, 55 N. Eagleville Road, Storrs, CT, 06268, USA. 7. Department of Biological Sciences and RNA Institute, University at Albany, 1400 Washington Avenue, Albany, NY, 12222, USA. mbelfort@albany.edu. 8. Department of Biomedical Sciences, School of Public Health, University at Albany, 1400 Washington Avenue, Albany, NY, 12222, USA. mbelfort@albany.edu. 9. Department of Biological Sciences and RNA Institute, University at Albany, 1400 Washington Avenue, Albany, NY, 12222, USA. novikoos@buffalostate.edu. 10. Current address: Biology Department, SUNY Buffalo State College, 1300 Elmwood Avenue, Buffalo, NY, 14222, USA. novikoos@buffalostate.edu.
Abstract
BACKGROUND: Group II introns are mobile retroelements, capable of invading new sites in DNA. They are self-splicing ribozymes that complex with an intron-encoded protein to form a ribonucleoprotein that targets DNA after splicing. These molecules can invade DNA site-specifically, through a process known as retrohoming, or can invade ectopic sites through retrotransposition. Retrotransposition, in particular, can be strongly influenced by both environmental and cellular factors. RESULTS: To investigate host factors that influence retrotransposition, we performed random insertional mutagenesis using the ISS1 transposon to generate a library of over 1000 mutants in Lactococcus lactis, the native host of the Ll.LtrB group II intron. By screening this library, we identified 92 mutants with increased retrotransposition frequencies (RTP-ups). We found that mutations in amino acid transport and metabolism tended to have increased retrotransposition frequencies. We further explored a subset of these RTP-up mutants, the most striking of which is a mutant in the ribosomal RNA methyltransferase rlmH, which exhibited a reproducible 20-fold increase in retrotransposition frequency. In vitro and in vivo experiments revealed that ribosomes in the rlmH mutant were defective in the m3Ψ modification and exhibited reduced binding to the intron RNA. CONCLUSIONS: Taken together, our results reinforce the importance of the native host organism in regulating group II intron retrotransposition. In particular, the evidence from the rlmH mutant suggests a role for ribosome modification in limiting rampant retrotransposition.
BACKGROUND: Group II introns are mobile retroelements, capable of invading new sites in DNA. They are self-splicing ribozymes that complex with an intron-encoded protein to form a ribonucleoprotein that targets DNA after splicing. These molecules can invade DNA site-specifically, through a process known as retrohoming, or can invade ectopic sites through retrotransposition. Retrotransposition, in particular, can be strongly influenced by both environmental and cellular factors. RESULTS: To investigate host factors that influence retrotransposition, we performed random insertional mutagenesis using the ISS1 transposon to generate a library of over 1000 mutants in Lactococcus lactis, the native host of the Ll.LtrB group II intron. By screening this library, we identified 92 mutants with increased retrotransposition frequencies (RTP-ups). We found that mutations in amino acid transport and metabolism tended to have increased retrotransposition frequencies. We further explored a subset of these RTP-up mutants, the most striking of which is a mutant in the ribosomal RNA methyltransferase rlmH, which exhibited a reproducible 20-fold increase in retrotransposition frequency. In vitro and in vivo experiments revealed that ribosomes in the rlmH mutant were defective in the m3Ψ modification and exhibited reduced binding to the intron RNA. CONCLUSIONS: Taken together, our results reinforce the importance of the native host organism in regulating group II intron retrotransposition. In particular, the evidence from the rlmH mutant suggests a role for ribosome modification in limiting rampant retrotransposition.
Entities:
Keywords:
Mobile genetic elements; RNA splicing; Retrotransposons; Ribosomes
Authors: Lydia M Contreras; Tao Huang; Carol Lyn Piazza; Dorie Smith; Guosheng Qu; Grant Gelderman; Jeffrey P Potratz; Rick Russell; Marlene Belfort Journal: RNA Date: 2013-09-17 Impact factor: 4.942