Involvement of DEAD-box Proteins in Group I and Group
II Intron Splicing. Biochemical Characterization of
Mss116p, ATP Hydrolysis-dependent and -independent
Mechanisms, and General RNA Chaperone Activity
Coralie Halls
1
†, Sabine Mohr
1
†, Mark Del Campo
1
†, Quansheng Yang
2
Eckhard Jankowsky
2
and Alan M. Lambowitz
1
⁎
1
Institute for Cellular and
Molecular Biology, Department
of Chemistry and Biochemistry,
and Section of Molecular,
Genetics and Microbiology,
School of Biological Sciences,
University of Texas at Austin,
Austin, Texas 78712, USA
2
Department of Biochemistry
and Center for RNA Molecular
Biology, Case Western Reserve
University, Cleveland,
OH 44106, USA
The RNA-catalyzed splicing of group I and group II introns is facilitated by
proteins that stabilize the active RNA structure or act as RNA chaperones to
disrupt stable inactive structures that are kinetic traps in RNA folding. In
Neurospora crassa and Saccharomyces cerevisiae, the latter function is fulfilled
by specific DEAD-box proteins, denoted CYT-19 and Mss116p, respectively.
Previous studies showed that purified CYT-19 stimulates the in vitro splicing
of structurally diverse group I and group II introns, and uses the energy of
ATP binding or hydrolysis to resolve kinetic traps. Here, we purified
Mss116p and show that it has RNA-dependent ATPase activity, unwinds
RNA duplexes in a non-polar fashion, and promotes ATP-independent
strand-annealing. Further, we show that Mss116p binds RNA non-
specifically and promotes in vitro splicing of both group I and group II
intron RNAs, as well as RNA cleavage by the aI5γ-derived D135 ribozyme.
However, Mss116p also has ATP hydrolysis-independent effects on some of
these reactions, which are not shared by CYT-19 and may reflect differences
in its RNA-binding properties. We also show that a non-mitochondrial
DEAD-box protein, yeast Ded1p, can function almost as efficiently as CYT-
19 and Mss116p in splicing the yeast aI5γ group II intron and less efficiently
in splicing the bI1 group II intron. Together, our results show that Mss116p,
like CYT-19, can act broadly as an RNA chaperone to stimulate the splicing
of diverse group I and group II introns, and that Ded1p also has an RNA
chaperone activity that can be assayed by its effect on splicing mitochon-
drial introns. Nevertheless, these DEAD-box protein RNA chaperones are
not completely interchangeable and appear to function in somewhat
different ways, using biochemical activities that have likely been tuned by
coevolution to function optimally on specific RNA substrates.
Published by Elsevier Ltd.
*Corresponding author
Keywords: catalytic RNA; ribozyme; RNA chaperone; RNA–protein
interaction; RNA structure
Introduction
Group I and group II introns splice via RNA-
catalyzed transesterification reactions that are facili-
tated by proteins.
1
Some of these proteins are RNA
splicing factors that bind specifically to the intron
RNA and stabilize the catalytically active RNA
structure, while others are RNA chaperones that
bind RNAs without apparent specificity and disrupt
stable inactive structures that are “kinetic traps”
during RNA folding. RNA splicing factors for group
I and group II introns include both intron-encoded
† C.H., S.M. and M.D. contributed equally to this
work.
Abbreviations used: mt, mitochondrial; PEI,
polyethylenenimine; SER, spliced-exon reopening; TEV,
tobacco etch virus.
E-mail address of the corresponding author:
lambowitz@mail.utexas.edu
doi:10.1016/j.jmb.2006.09.083 J. Mol. Biol. (2007) 365, 835–855
0022-2836/$ - see front matter. Published by Elsevier Ltd.