The <i>Neurospora crassa</i> CYT-18 protein C-terminal RNA-binding domain helps stabilize interdomain tertiary interactions in group I introns

Chen, Xin; Mohr, Georg; Lambowitz, Alan M.

doi:10.1261/rna.5212604

Cited by 18 publications

(27 citation statements)

References 35 publications

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“…To account for cleavages in P5 on the opposite end of the intron RNA, the C-terminal domain of the other subunit (Subunit A) must be located in proximity to the L9-P5 tertiary interaction. These locations explain previous findings that CYT-18’s C-terminal domain can suppress mutations that weaken both the L2-P8 and L9-P5 tertiary interactions (15) and that small deletions in Ins2, which helps orient P9, have more severe effects in the C-terminally truncated than in the full-length protein (17). The C-terminal domain can also compensate for mutations in several other regions of the intron that may indirectly affect the relative orientation of the core domains or positioning of P2 or P8 (15).…”

Section: Discussionsupporting

confidence: 79%

“…Mt TyrRSs are structurally homologous to the bacterial enzymes, and CYT-18 uses both its N-terminal catalytic and C-terminal tRNA-binding domains to bind group I intron RNAs (13–15). However, group I intron splicing activity has been found only for the mt TyrRSs of Pezizomycotina, filamentous fungi that includes the model organisms Neurospora crassa , Aspergillus nidulans , and Podospora anserina , as well as important human pathogens, such as Histoplasma capsulatum, Coccidioides posadasii , and Aspergillus fumigatus (2).…”

mentioning

confidence: 99%

“…Although a C-terminally truncated CYT-18 protein consisting of the N-terminal catalytic and intermediate α-helical domains can splice many group I introns, the C-terminal domain contributes to group I intron binding and is essential for splicing some introns, e.g ., the N. crassa mt large ribosomal subunit (Nc mt LSU) intron (14). Genetic studies showed that CYT-18’s C-terminal domain is needed to compensate for certain structural mutations that impair self-splicing of group I intron RNAs, including mutations that weaken two key long-range tertiary interactions: L9-P5, which helps establish the correct relative orientation of the two catalytic core domains, and L2-P8, which helps position the P1 helix containing the 5’-splice site at the RNA’s active site (15). Additionally, small deletions within the catalytic domain’s Ins2, which binds near the L9-P5 tetraloop-tetraloop receptor interaction, have more severe effects in the C-terminally truncated CYT-18/Δ424–669 protein than in the full-length protein, suggesting that C-terminal domain binding can compensate for loss of some N-terminal domain interactions (17).…”

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confidence: 99%

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NMR Structure of the C-Terminal Domain of a Tyrosyl-tRNA Synthetase That Functions in Group I Intron Splicing

et al. 2011

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The mitochondrial tyrosyl-tRNA synthetases (mt TyrRSs) of Pezizomycotina fungi are bifunctional proteins that aminoacylate mitochondrial tRNATyr and are structure-stabilizing splicing cofactors for group I introns. Studies with the Neurospora crassa synthetase (CYT-18 protein) showed that splicing activity is dependent upon Pezizomycotina-specific structural adaptations that form a distinct group I intron-binding site in the N-terminal catalytic domain. Although CYT-18’s C-terminal domain also binds group I introns, it has been intractable to X-ray crystallography in the full-length protein. Here, we determined an NMR structure of the isolated C-terminal domain of the Aspergillus nidulans mt TyrRS, which is closely related to but smaller than CYT-18’s. The structure shows an S4 fold like that of bacterial TyrRSs, but with novel features, including three Pezizomycontia-specific insertions. 15N-1H two-dimensional NMR showed that C-terminal domains of the full-length A. nidulans and GeoBacillus stearothermophilus synthetases do not tumble independently in solution, suggesting restricted orientations. Modeling onto a CYT-18/group I intron co-crystal structure indicates that the C-terminal domains of both subunits of the homodimeric protein bind different ends of the intron RNA, with one C-terminal domain having to undergo a large shift on its flexible linker to bind tRNATyr or the intron RNA on either side of the catalytic domain. The modeling suggests that the C-terminal domain acts together with the N-terminal domain to clamp parts of the intron’s catalytic core, that at least one C-terminal domain insertion functions in group I intron binding, and that some C-terminal domain regions bind both tRNATyr and group I intron RNAs.

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Section: Discussionsupporting

confidence: 79%

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confidence: 99%

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confidence: 99%

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NMR Structure of the C-Terminal Domain of a Tyrosyl-tRNA Synthetase That Functions in Group I Intron Splicing

et al. 2011

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“…The mitochondrial tyrosyl-tRNA synthetase CYT-18 from N. crassa is a bifunctional protein, which, in addition to aminoacylation of its cognate tRNA, has also adapted to facilitate folding of N. crassa mitochondrial group I introns via stabilization of the RNA tertiary interactions 47; 48; 49. Although biochemical studies indicated that the protein recognizes a tRNA-like structure within group I introns 47, recent crystallographic studies indicate that the protein uses completely different binding surfaces to interact with tRNA and group I intron RNAs 49.…”

Section: Discussionmentioning

confidence: 99%

Protein-Facilitated Folding of Group II Intron Ribozymes

Fedorova

Solem

Pyle

2010

Journal of Molecular Biology

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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.

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“…Like its bacterial counterparts, CYT-18 functions as a homodimer, with each dimer binding either one molecule of tRNA Tyr or group I intron RNA [25] – [28] . CYT-18 binds group I introns by using both its N-terminal catalytic domain and CTD, but only some introns require the CTD for RNA splicing [29] – [31] .…”

Section: Introductionmentioning

confidence: 99%

Evolution of RNA-Protein Interactions: Non-Specific Binding Led to RNA Splicing Activity of Fungal Mitochondrial Tyrosyl-tRNA Synthetases

2014

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The Neurospora crassa CYT-18 protein C-terminal RNA-binding domain helps stabilize interdomain tertiary interactions in group I introns

Cited by 18 publications

References 35 publications

NMR Structure of the C-Terminal Domain of a Tyrosyl-tRNA Synthetase That Functions in Group I Intron Splicing

NMR Structure of the C-Terminal Domain of a Tyrosyl-tRNA Synthetase That Functions in Group I Intron Splicing

Protein-Facilitated Folding of Group II Intron Ribozymes

Evolution of RNA-Protein Interactions: Non-Specific Binding Led to RNA Splicing Activity of Fungal Mitochondrial Tyrosyl-tRNA Synthetases

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