Four KH domains of the <i>C. elegans</i> Bicaudal-C ortholog GLD-3 form a globular structural platform

Nakel, Katharina; Hartung, Sophia; Bonneau, Fabien; Eckmann, Christian R.; Conti, Elena

doi:10.1261/rna.2315010

Cited by 27 publications

(35 citation statements)

References 34 publications

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“…Hollingworth et al reported that a GDDG double mutation in KH-type splicing regulatory protein (KSRP) impairs nucleic acid binding without compromising KH domain stability (40). The absence or augmentation of the canonical GxxG sequence appears to be a hallmark of impairment of RNA binding (41,42). Our data provide further support for this hypothesis.…”

Section: Discussionsupporting

confidence: 78%

S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation

et al. 2013

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To better understand the roles of the KH and S1 domains in RNA binding and polynucleotide phosphorylase (PNPase) autoregulation, we have identified and investigated key residues in these domains. A convenient pnp::lacZ fusion reporter strain was used to assess autoregulation by mutant PNPase proteins lacking the KH and/or S1 domains or containing point mutations in those domains. Mutant enzymes were purified and studied by using in vitro band shift and phosphorolysis assays to gauge binding and enzymatic activity. We show that reductions in substrate affinity accompany impairment of PNPase autoregulation. A remarkably strong correlation was observed between ␤-galactosidase levels reflecting autoregulation and apparent K D values for the binding of a model RNA substrate. These data show that both the KH and S1 domains of PNPase play critical roles in substrate binding and autoregulation. The findings are discussed in the context of the structure, binding sites, and function of PNPase. Polynucleotide phosphorylase (PNPase) is a conserved, widely distributed phosphorolytic 3=-5= exoribonuclease that may also function under some circumstances as a template-independent RNA polymerase (1; reviewed in reference 2). Although it is not essential, deletions of its gene (pnp) are synthetically lethal when either RNase II or RNase R, both of which are hydrolytic 3=-5= exoribonucleases, is deficient (3-5). Strains deficient in PNPase are also sensitive to cold shock and other stresses (6-9). Thus, PNPase is believed to play significant roles in mRNA turnover and other aspects of RNA processing and metabolism (4, 10-12). Partial or full structures of PNPase from several microorganisms (13-16), as well as structures of the related archaeal exosome (17, 18), have shed considerable light on its mechanism of action. Bacterial PNPase is composed of three identical subunits. Each subunit consists of two tandem globular domains (residues 8 to 210 and 312 to 541; see Fig. 1a) derived from RNase PH that form a core whose central channel is accessible from both the upper and lower surfaces of the core (14,19). Each subunit also contains a C-terminal extension that consists of two additional small domains, KH and S1 (residues 551 to 591 and 622 to 691, respectively; Fig. 1), which are positioned on the upper surface of the core. The KH and S1 domains have been implicated in autoregulation (20), in resistance to cold shock (7), in pathogenesis (21), and in substrate binding (22). Although a solution structure of the S1 domain from Escherichia coli PNPase has been available (13), the positions of the S1 and KH domains relative to the core have been elucidated only recently from the structure of PNPase from Caulobacter crescentus (16). The S1 and KH domains do not appear to be required for the enzymatic activity of PNPase (20,22,23). Nonetheless, deletion of the S1, the KH or both domains results in significant loss of RNA binding and inefficient enzymatic turnover (22).PNPase from E. coli and other organisms exhibits strong autoregulation...

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Section: Discussionsupporting

confidence: 78%

S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation

et al. 2013

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“…Three-dimensional structural analysis has revealed that all fi ve KH domains of GLD-3 have extensive contacts with each other, forming a tightly packaged protein core, which is in contrast to a previously assumed "beads on a string" organization. Consistent with GLD-3 having an extremely low af fi nity for homopolymeric guanidine RNA (Jedamzik and Eckmann, unpublished results), the typical GxxG RNA-contacting loops (G, glycine) of known RNAbinding KH domains are missing either one or both of the two glycine residues (Nakel et al 2010 ) . GLD-3 binds GLD-2, and GLS-1 (see below).…”

Section: Kh Proteinssupporting

confidence: 55%

“…They contain 5 KH domains arranged in tandem connected via very short amino acid linkers, whereby each individual domain adopts a classic KH fold (Eckmann et al 2002 ;Nakel et al 2010 ) . Three-dimensional structural analysis has revealed that all fi ve KH domains of GLD-3 have extensive contacts with each other, forming a tightly packaged protein core, which is in contrast to a previously assumed "beads on a string" organization.…”

Section: Kh Proteinsmentioning

confidence: 99%

Translational Control in the Caenorhabditis elegans Germ Line

Nousch

Eckmann

2012

Advances in Experimental Medicine and Biology

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Translational control is a prevalent form of gene expression regulation in the Caenorhabditis elegans germ line. Linking the amount of protein synthesis to mRNA quantity and translational accessibility in the cell cytoplasm provides unique advantages over DNA-based controls for developing germ cells. This mode of gene expression is especially exploited in germ cell fate decisions and during oogenesis, when the developing oocytes stockpile hundreds of different mRNAs required for early embryogenesis. Consequently, a dense web of RNA regulators, consisting of diverse RNA-binding proteins and RNA-modifying enzymes, control the translatability of entire mRNA expression programs. These RNA regulatory networks are tightly coupled to germ cell developmental progression and are themselves under translational control. The underlying molecular mechanisms and RNA codes embedded in the mRNA molecules are beginning to be understood. Hence, the C. elegans germ line offers fertile grounds for discovering post-transcriptional mRNA regulatory mechanisms and emerges as great model for a systems level understanding of translational control during development.

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“…KH-domains that do not contain the conserved GxxG motif -and do not interact with RNA -can assemble in multi-domain units where more than one KH-KH interface is observed, exemplified by the structure of the type I KH tetra-domain unit of Bicaudal-C (Bic-C) (Figure 4b) [39]. Type II KH domains lacking the GxxG motif have also been recently reported in proteins of the metallo-β-lactamase superfamily.…”

Section: Combinatorial Binding and Architectural Role By Multiple Kh mentioning

confidence: 84%

KH–RNA interactions: back in the groove

Nicastro¹,

Taylor²,

Ramos

2015

Current Opinion in Structural Biology

125

131

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The hnRNP K-homology (KH) domain is a single stranded nucleic acid binding domain that mediates RNA target recognition by a large group of gene regulators. The structure of the KH fold is well characterised and some initial rules for KH-RNA recognition have been drafted. However, recent findings have shown that these rules need to be revisited and have now provided a better understanding of how the domain can recognize a sequence landscape larger than previously thought as well as revealing the diversity of structural expansions to the KH domain. Finally, novel structural and functional data show how multiple KH domains act in a combinatorial fashion to both allow recognition of longer RNA motifs and remodelling of the RNA structure. These advances set the scene for a detailed molecular understanding of KH selection of the cellular targets.

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Four KH domains of the C. elegans Bicaudal-C ortholog GLD-3 form a globular structural platform

Cited by 27 publications

References 34 publications

S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation

S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation

Translational Control in the Caenorhabditis elegans Germ Line

KH–RNA interactions: back in the groove

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