Do DEAD-Box Proteins Promote Group II Intron Splicing without Unwinding RNA?

DEAD-box proteins are ubiquitous in RNA metabolism and use ATP to mediate RNA conformational changes. These proteins have been suggested to use a fundamentally different mechanism from the related DNA and RNA helicases, generating local strand separation while remaining tethered through additional interactions with structured RNAs and RNA-protein (RNP) complexes. Here, we provide a critical test of this model by measuring the number of ATP molecules hydrolyzed by DEAD-box proteins as they separate short RNA helices characteristic of structured RNAs (6 -11 bp). We show that the DEAD-box protein CYT-19 can achieve complete strand separation using a single ATP, and that 2 related proteins, Mss116p and Ded1p, display similar behavior. Under some conditions, considerably <1 ATP is hydrolyzed per separation event, even though strand separation is strongly dependent on ATP and is not supported by the nucleotide analog AMP-PNP. Thus, ATP strongly enhances strand separation activity even without being hydrolyzed, most likely by eliciting or stabilizing a protein conformation that promotes strand separation, and AMP-PNP does not mimic ATP in this regard. Together, our results show that DEADbox proteins can disrupt short duplexes by using a single cycle of ATP-dependent conformational changes, strongly supporting and extending models in which DEAD-box proteins perform local rearrangements while remaining tethered to their target RNAs or RNP complexes. This mechanism may underlie the functions of DEAD-box proteins by allowing them to generate local rearrangements without disrupting the global structures of their targets.CYT-19 ͉ group I intron ͉ RNA chaperone ͉ RNA folding ͉ RNA helicase S tructured RNAs and RNA-protein complexes (RNPs) mediate a host of essential cellular processes, including processing of messenger RNAs and their translation into protein. In addition to folding into defined structures, many of these RNAs and RNPs undergo extensive conformational changes during their functions. Both their initial folding and conformational changes typically require DEAD-box proteins, which use ATP to promote RNA structural transitions.DEAD-box proteins are members of helicase superfamily-2 (SF2) and are related to the ATP-dependent RNA and DNA helicases that function in replication and other aspects of nucleic acid metabolism (1). However, rather than unwinding long, continuous duplexes, many DEAD-box proteins manipulate highly structured RNAs and RNPs by facilitating rearrangements that can include local disruptions of secondary structure, tertiary structure, and RNA-protein interactions.Consistent with their distinct functions, recent in vitro studies have strongly suggested that DEAD-box proteins operate on structured RNAs by a mechanism that is fundamentally different from processive helicases. The Neurospora crassa CYT-19 protein is required for proper folding of several mitochondrial group I introns in Neurospora crassa (2). It also assists folding of diverse group I and group II introns in vitro or when expressed in...

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“…S3). This activity may be achieved by ''strand capture,'' analogous to the RNA chaperone activity of proteins that are not ATPases (18)(19)(20).…”

Section: Resultsmentioning

confidence: 99%

“…AMP-PNP was treated to remove any contaminating ATP as described in ref. 18. RNA and nucleotide concentrations were determined spectrophotometrically (see SI Text, Determination of RNA and Nucleotide Concentrations).…”

Section: Methodsmentioning

confidence: 99%

DEAD-box proteins can completely separate an RNA duplex using a single ATP

Chen

Potratz²,

Tijerina³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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148

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“…Mss116 may also contribute to the folding of the aI5␤ intron. Mss116 was shown recently to stabilize the tertiary fold of the aI5␥ intron (14,15). Mne1 may also contribute to this stabilization function through its ability to bind COX1 mRNA, although its interaction is not exclusively via the aI5␤ intron.…”

Section: Discussionmentioning

confidence: 99%

“…Some of the nuclear factors, e.g. Mss116, have been shown to facilitate the RNA folding reaction or stabilize the RNA tertiary fold (14,15).COX1 introns in yeast are annotated as aI1-aI4 and aI5␣, -␤, and -␥. The aI3, aI4, aI5␣, and aI5␤ introns are group I introns, whereas aI1, aI2, and aI5␥ are group II introns.…”

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confidence: 99%

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Mne1 Is a Novel Component of the Mitochondrial Splicing Apparatus Responsible for Processing of a COX1 Group I Intron in Yeast

Watts

Khalimonchuk

Wolf

et al. 2011

Journal of Biological Chemistry

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Saccharomyces cerevisiae cells lacking Mne1 are deficient in intron splicing in the gene encoding the Cox1 subunit of cytochrome oxidase but contain wild-type levels of the bc 1 complex. Thus, Mne1 has no role in splicing of COB introns or expression of the COB gene. Northern experiments suggest that splicing of the COX1 aI5␤ intron is dependent on Mne1 in addition to the previously known Mrs1, Mss116, Pet54, and Suv3 factors. Processing of the aI5␤ intron is similarly impaired in mne1⌬ and mrs1⌬ cells and overexpression of Mrs1 partially restores the respiratory function of mne1⌬ cells. Mrs1 is known to function in the initial transesterification reaction of splicing. Mne1 is a mitochondrial matrix protein loosely associated with the inner membrane and is found in a high mass ribonucleoprotein complex specifically associated with the COX1 mRNA even within an intronless strain. Mne1 does not appear to have a secondary function in COX1 processing or translation, because disruption of MNE1 in cells containing intronless mtDNA does not lead to a respiratory growth defect. Thus, the primary defect in mne1⌬ cells is splicing of the aI5␤ intron in COX1.Cytochrome c oxidase (CcO) 3 biogenesis requires the expression and interaction of subunits encoded by mitochondrial and nuclear genomes. A myriad of nuclear-encoded assembly factors mediate CcO biogenesis (1, 2). These factors function in the processing of mitochondrial CcO subunit mRNAs, their translation and insertion into the inner membrane, and formation of metal and heme cofactor centers. Assembly is initiated with the synthesis and maturation of the Cox1 subunit, one of the three catalytic core components (3, 4). COX1 is the one CcO gene that is universally present in the mitochondrial genome. The COX1 mRNA requires processing prior to translation on mitochondrial ribosomes. Cox1 contains three of the redox centers of the enzyme with heme a, a 3 , and Cu B cofactors (5). The last redox center, the binuclear Cu A center, exists within the Cox2 subunit.Mitochondrial genomes of non-metazoan species tend to be larger due to the presence of non-coding regions including introns and additional genes not present in animals (6 -8). In Saccharomyces cerevisiae, introns are commonly found in COX1 as well as COB (the cytochrome b subunit of the bc 1 complex) and the large ribosomal RNA gene (9). Two types of introns, groups I and II, exist in COX1. Some of these introns contain open reading frames encoding maturases, related to DNA endonucleases for group I introns and reverse transcriptases for group II introns (8). Group I intron-containing COX1 alleles have been generated numerous times during evolution due to the invasive nature of the introns containing a DNA homing endonuclease (10). Most commonly used laboratory yeast strains contain up to seven introns within COX1 and in such multiple-intron strains, exons may be as short as 24 -37 bases. No compelling rationale is known why introns are maintained in COX1 and COB.The generation of mature COX1 mRNA transcript requir...

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DEAD‐box proteins as RNA helicases and chaperones

2010

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DEAD-box proteins are ubiquitous in RNA-mediated processes and function by coupling cycles of ATP binding and hydrolysis to changes in affinity for single-stranded RNA. Many DEAD-box proteins use this basic mechanism as the foundation for a version of RNA helicase activity, efficiently separating the strands of short RNA duplexes in a process that involves little or no translocation. This activity, coupled with mechanisms to direct different DEAD-box proteins to their physiological substrates, allows them to promote RNA folding steps and rearrangements and to accelerate remodeling of RNA-protein complexes. This review will describe the properties of DEAD-box proteins as RNA helicases and the current understanding of how the energy from ATPase activity is used to drive the separation of RNA duplex strands. It will then describe how the basic biochemical properties allow some DEAD-box proteins to function as chaperones by promoting RNA folding reactions, with a focus on the self-splicing group I and group II intron RNAs. KeywordsATPase; CYT-19; DExD/H-box protein; Group I intron; Group II intron; Mss116; RNA folding DEAD-box proteins are the largest family of superfamily 2 (SF2) helicases. They share with other SF2 proteins a helicase core composed of two RecA-like domains and a common set of sequence motifs. In the DEAD-box proteins, motif II includes the sequence D-E-A-D, which is the origin of their name (1). DEAD-box proteins are found in all three domains of life and perform a diverse set of functions in an even more diverse set of cellular processes, including assembly and function of macromolecular machines like the ribosome and spliceosome, quality control mechanisms in gene expression, nuclear export of mRNA, folding of self-splicing RNA introns, and others (2,3) ( Table 1). The broader term "DExD/ H-box" has been used to include other proteins with related motif II sequences (2,3), but a recent comparative sequence analysis has led to questions as to the relevance of this designation, because DEAD-box proteins are not more closely related to DExH helicases than to other families of SF2 helicases (4).In keeping with the broad range of functions for DEAD-box proteins, there is also considerable diversity in their mechanisms. Nevertheless, common themes are emerging. Since their discovery more than 20 years ago (1), the central defining biochemical activity of DEAD-box proteins has been their ability to bind and hydrolyze ATP in a cycle that is stimulated by binding to single-stranded or double-stranded RNA (ssRNA or dsRNA). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptSubsequent work has shown that the energetic communication implied by the stimulation of ATPase activity by RNA binding forms a common set of properties that underlies the diverse mechanisms of DEAD-box proteins. Progression through the ATPase cycle leads to changes in ssRNA affinity, allowing DEAD-box proteins to bind and release RNA in a regulated manner. This regulated binding is then used by different ...

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Do DEAD-Box Proteins Promote Group II Intron Splicing without Unwinding RNA?

Cited by 59 publications

References 21 publications

DEAD-box proteins can completely separate an RNA duplex using a single ATP

DEAD-box proteins can completely separate an RNA duplex using a single ATP

Mne1 Is a Novel Component of the Mitochondrial Splicing Apparatus Responsible for Processing of a COX1 Group I Intron in Yeast

DEAD‐box proteins as RNA helicases and chaperones

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