KH domains with impaired nucleic acid binding as a tool for functional analysis

Hollingworth, David; Candel, Adela M.; Nicastro, Giuseppe; Martin, Stephen R.; Briata, Paola; Gherzi, Roberto; Ramos, Andrés

doi:10.1093/nar/gks368

Cited by 118 publications

(107 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…4C and Fig. S4D, the interaction of KSRP mutants with transfected myogenin 3′UTR or endogenous KSRP mRNA targets (GNAS and CTNNB1, also known as β-catenin) occurred according to the previously described mode with KH2, KH3, and KH4 playing the major role in the RNA recognition and KH1 resulting dispensable (28,29). The interaction of KSRP with H19 was unaffected by mutations in KH4 but was impaired not only by mutations in KH2 and 3 but also in KH1 (Fig.…”

Section: H19 Silencing In Undifferentiated C2c12 Cells Promotes Myogementioning

confidence: 65%

“…All plasmids were sequenced before their use. pcDNA3-Flag-KSRP, pCMV-TAG2B-KSRP, pCMV-TAG2B-KSRP(S193D), pCMV-TAG2B-KH1GDDG, pCMV-TAG2B-KH2GDDG, pCMV-TAG2B-KH3GDDG, pCMV-TAG2B-KH4GDDG, pcDNA-3-myrAKT2, pcDNA3-MKK6EE, pcDNA3-EXOSC2, and pcDNA3-EXOSC5 plasmids were described elsewhere (13,14,21,28,46). Adenoviral vector expressing myristoylated AKT2 and the respective negative control were from Vector Biolabs.…”

Section: Methodsmentioning

confidence: 99%

“…We have recently generated a series of KSRP mutants in which the hallmark GxxG RNA binding loop present in each KH domain is substituted by the GDDG sequence (28). Thus, we obtained four KSRP mutants in which the RNA binding ability of each single KH domain is abrogated whereas the structure and stability of the protein is unaffected (KH1GDDG, KH2GDDG, KH3GDDG, KH4GDDG) (28). We transiently expressed each KSRP mutant (or WT KSRP) in HEK-293 cells (Fig.…”

Section: H19 Silencing In Undifferentiated C2c12 Cells Promotes Myogementioning

confidence: 99%

See 2 more Smart Citations

H19 long noncoding RNA controls the mRNA decay promoting function of KSRP

Giovarelli

Bucci

Ramos

et al. 2014

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

Self Cite

107

View full text Add to dashboard Cite

Long noncoding RNAs (lncRNAs) interact with protein factors to regulate different layers of gene expression transcriptionally or posttranscriptionally. Here we report on the functional consequences of the unanticipated interaction of the RNA binding protein K homology-type splicing regulatory protein (KSRP) with the H19 lncRNA (H19). KSRP directly binds to H19 in the cytoplasm of undifferentiated multipotent mesenchymal C2C12 cells, and this interaction favors KSRP-mediated destabilization of labile transcripts such as myogenin. AKT activation induces KSRP dismissal from H19 and, as a consequence, myogenin mRNA is stabilized while KSRP is repurposed to promote maturation of myogenic microRNAs, thus favoring myogenic differentiation. Our data indicate that H19 operates as a molecular scaffold that facilitates effective association of KSRP with myogenin and other labile transcripts, and we propose that H19 works with KSRP to optimize an AKT-regulated posttranscriptional switch that controls myogenic differentiation. Among small noncoding RNAs, microRNAs (miRNAs) repress gene expression by inhibiting translation and/or promoting decay of target mRNAs (1), with RBPs being able to control miRNA maturation from precursors or influence miRNA function (1, 2). A new class of transcripts referred to as long noncoding RNAs (lncRNAs, arbitrarily defined as longer than 200 nt) has recently moved to the forefront of regulatory RNA research (3). lncRNAs had been originally considered epigenetic regulators of gene expression, but the emphasis placed on the ways they regulate chromatin state likely obscures the full repertoire of their functions (4-6). Other roles of lncRNAs include posttranscriptional regulation, posttranslational control of protein activity, organization of protein complexes, as well as cell-cell signaling (4, 6). lncRNAs have been implicated in cellular events as different as cell cycle regulation, pluripotency, apoptosis, and DNA damage response, to name just a few (5, 6). Not surprisingly, lncRNA expression is altered in cancer, and it is becoming clear that some lncRNAs can control cell transformation by regulating vital cellular functions (7). Nevertheless, the composition and function of ribonucleoprotein complexes, including lncRNAs, is generally uncharacterized.Studies performed in primary and cultured cells as well as in mice proved that the K homology (KH)-type splicing regulatory protein (KSRP), a single-stranded RBP that interacts with nucleic acids through four hnRNP KH domains, is able to integrate different levels of gene expression and is required for proper immune response, lipid metabolism, cell fate decisions, and tissue regeneration (see ref. 8 for a recent review). We and others have found that KSRP negatively regulates gene expression via at least two distinct and integrated posttranscriptional mechanisms: (i) by promoting decay of unstable mRNAs [mainly targeting AU-rich elements (AREs) in their 3′UTRs] (8, 9) and (ii) by favoring maturation of select miRNAs from precursors (8, 10). Brie...

show abstract

Section: H19 Silencing In Undifferentiated C2c12 Cells Promotes Myogementioning

confidence: 65%

Section: Methodsmentioning

confidence: 99%

Section: H19 Silencing In Undifferentiated C2c12 Cells Promotes Myogementioning

confidence: 99%

See 1 more Smart Citation

H19 long noncoding RNA controls the mRNA decay promoting function of KSRP

Giovarelli

Bucci

Ramos

et al. 2014

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

Self Cite

107

View full text Add to dashboard Cite

show abstract

“…Although the effects were less pronounced, both K571L and K571Q also resulted in losses of repression in vivo and weakened K D values in vitro. 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).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2013

View full text Add to dashboard Cite

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...

show abstract

“…Correspondingly, the RNA binding function of the KH domains of NusA, GLD-1, Sam68, and heterogeneous nuclear RNP K were all severely impeded upon mutating the first glycine in the GXXG motif to an aspartate (24 -27). In fact, a recent study showed the feasibility of double mutating the GXXG loop, namely from GXXG to GDDG, in the KH domain of the RNAbinding protein KSRP and the KH3 and KH4 domains of IMP1, as an effective tool for the investigation of the nucleic acidbinding function of individual KH domains (28).…”

Section: The Ability Of Its Four Heterogeneous Nuclear Rnp-k-homologymentioning

confidence: 99%

Molecular Insights into the Coding Region Determinant-binding Protein-RNA Interaction through Site-directed Mutagenesis in the Heterogeneous Nuclear Ribonucleoprotein-K-homology Domains

Barnes

Rensburg

et al. 2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: Coding region determinant-binding protein (CRD-BP) interacts physically with oncogenic mRNAs. Results: Point mutation in the K-homology (KH) domains of CRD-BP abolishes its RNA-binding ability. Conclusion: Two KH domains of CRD-BP are required for efficient binding to oncogenic mRNAs and for granule formation in zebrafish embryos. Significance: Learning how the KH domains interact with mRNAs is crucial for understanding oncogenic function of CRD-BP.

show abstract

KH domains with impaired nucleic acid binding as a tool for functional analysis

Cited by 118 publications

References 42 publications

H19 long noncoding RNA controls the mRNA decay promoting function of KSRP

H19 long noncoding RNA controls the mRNA decay promoting function of KSRP

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

Molecular Insights into the Coding Region Determinant-binding Protein-RNA Interaction through Site-directed Mutagenesis in the Heterogeneous Nuclear Ribonucleoprotein-K-homology Domains

Contact Info

Product

Resources

About