Protein-Facilitated Folding of Group II Intron Ribozymes

Abstract

Multiple studies hypothesize that DEAD-box proteins facilitate folding of the ai5γ group II intron. However, these conclusions are generally inferred from splicing kinetics, and not from direct monitoring of DEAD-box protein-facilitated folding of the intron. Using native gel electrophoresis and DMS structural probing, we monitored Mss 116-facilitated folding of ai5γ intron ribozymes and a catalytically active self-splicing RNA containing full length intron and short exons. We found that the protein directly stimulates folding of these RNAs by accelerating formation of the compact near-native state. This process occurs in an ATP-independent manner, although, ATP is required for the protein turnover. As Mss 116 binds RNA non-specifically, most of binding events do not result in the formation of the compact state, and ATP is required for the protein to dissociate from such non-productive complexes and rebind the unfolded RNA. Results obtained from experiments at different concentrations of magnesium ions suggest that Mss 116 stimulates folding of ai5γ ribozymes by promoting the formation of unstable folding intermediates, which is then followed by a cascade of folding events resulting in the formation of the compact near-native state. DMS probing results suggest that the compact state formed in the presence of the protein is identical to the near-native state formed more slowly in its absence. Our results also indicate that Mss 116 does not stabilize the native state of the ribozyme, but that such stabilization results from binding of attached exons.

From unwinding to clamping — the DEAD box RNA helicase family

“…Although we concentrate in this Review on only three instructive examples, it is important to note that there are additional ways in which DEAD box proteins can regulate RNAdependent processes. For example, significant attention has been focused on several fungal mitochondrial DEAD box helicases, including Mss116 and CYT19, which act as RNA chaperones to promote folding of several mitochondrial RNA introns into their native conformations 45,57,61,[127][128][129][130] . For this function, it seems critical that the DEAD box proteins bind and affect RNA structure in a non-sequence-or structure-specific fashion, much like the way in which traditional protein chaperones act on proteins 6 .…”

RNA folding in living cells

“…92 While the early information on Mss116p's mechanism was entirely inferred from splicing kinetics, Pyle and coworkers recently directly monitored DEAD-box protein-facilitated folding of the ai5γ intron. 97 Mss116p was observed to directly stimulate ai5γ folding by accelerating the collapse to the near-native state in an ATP-independent manner through stabilization of an early folding intermediate. 97 ATP on the other hand is required for the of action has not been deciphered yet.…”

“…97 Mss116p was observed to directly stimulate ai5γ folding by accelerating the collapse to the near-native state in an ATP-independent manner through stabilization of an early folding intermediate. 97 ATP on the other hand is required for the of action has not been deciphered yet. It is possible that there are different modes underlying RNA chaperone activity.…”

DEAD-box protein facilitated RNA folding in vivo

“…14,15,41 In vitro the ai5γ intron requires non-physiological ribozyme appears to be stabilized by binding of attached exons but not by Mss116p. 33 At the same time, Mss116p influences the folding mechanism in another way: long exon sequences interfere with ai5γ splicing in vitro. 34 Apparently, the unwinding activity of Mss116p is essential for exon unfolding, but not for intron folding in vitro.…”

“…25,27,28,32 Most recently, it has been shown that Mss116p stimulates ai5γ folding in vitro by accelerating the collapse to a near-native, compact intermediate state in an ATPindependent manner through stabilization of an early, unstable folding intermediate. 33 Interestingly, the native state of the ai5γ ©2 0 1 1 L a n d e s B i o s c i e n c e .…”