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The STAR RNA binding proteins GLD-1, QKI, SAM68 and SLM-2 bind bipartite RNA motifs.

Galarneau A, Richard S - BMC Mol. Biol. (2009)

Bottom Line: The GLD-1 binding site is defined as a hexanucleotide sequence (NACUCA) that is found in many, but not all, physiological GLD-1 mRNA targets.We also confirmed that GLD-1 also binds a bipartite RNA sequence in vitro with a short RNA sequence from its tra-2 physiological mRNA target.This information should help identify binding sites within physiological RNA targets.

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Affiliation: Bloomfield Center for Research on Aging, Lady Davis Institute for Medical Research, Sir Mortimer B, Davis Jewish General Hospital, and Department of Oncology, McGill University, Montréal, Québec, Canada. andre.galarneau@spcorp.com

ABSTRACT

Background: SAM68, SAM68-like mammalian protein 1 (SLM-1) and 2 (SLM-2) are members of the K homology (KH) and STAR (signal transduction activator of RNA metabolism) protein family. The function of these RNA binding proteins has been difficult to elucidate mainly because of lack of genetic data providing insights about their physiological RNA targets. In comparison, genetic studies in mice and C. elegans have provided evidence as to the physiological mRNA targets of QUAKING and GLD-1 proteins, two other members of the STAR protein family. The GLD-1 binding site is defined as a hexanucleotide sequence (NACUCA) that is found in many, but not all, physiological GLD-1 mRNA targets. Previously by using Systematic Evolution of Ligands by EXponential enrichment (SELEX), we defined the QUAKING binding site as a hexanucleotide sequence with an additional half-site (UAAY). This sequence was identified in QKI mRNA targets including the mRNAs for myelin basic proteins.

Results: Herein we report using SELEX the identification of the SLM-2 RNA binding site as direct U(U/A)AA repeats. The bipartite nature of the consensus sequence was essential for SLM-2 high affinity RNA binding. The identification of a bipartite mRNA binding site for QKI and now SLM-2 prompted us to determine whether SAM68 and GLD-1 also bind bipartite direct repeats. Indeed SAM68 bound the SLM-2 consensus and required both U(U/A)AA motifs. We also confirmed that GLD-1 also binds a bipartite RNA sequence in vitro with a short RNA sequence from its tra-2 physiological mRNA target.

Conclusion: These data demonstrate that the STAR proteins QKI, GLD-1, SAM68 and SLM-2 recognize RNA with direct repeats as bipartite motifs. This information should help identify binding sites within physiological RNA targets.

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GLD-1 binding to the tra2/gli element analysis. (A) RNA species tested in (B) and (C) are shown. The black bars denote sequences that are unaltered between the wild-type and the mutant versions. EMSAs of the tra2/Gli element with increasing concentrations of GLD-1 (B) and QKI (C) (by a factor of 2 from 2 nM) or with buffer alone. Migration patterns of unbound RNAs (free probe) and protein/bound RNAs (GLD-1/RNA or QKI/RNA complexes) are indicated on the left.
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Figure 4: GLD-1 binding to the tra2/gli element analysis. (A) RNA species tested in (B) and (C) are shown. The black bars denote sequences that are unaltered between the wild-type and the mutant versions. EMSAs of the tra2/Gli element with increasing concentrations of GLD-1 (B) and QKI (C) (by a factor of 2 from 2 nM) or with buffer alone. Migration patterns of unbound RNAs (free probe) and protein/bound RNAs (GLD-1/RNA or QKI/RNA complexes) are indicated on the left.

Mentions: A high affinity RNA binding site has been defined for C. elegans GLD-1 that consists of a hexanucleotide (NACU(C/A)A) [37]. To examine whether the GLD-1 hexanucleotide sequence also requires a similar half-site, we performed EMSA assays with a segment of the tra2 and gli repeated element (TGE) containing the hexanucleotide (UACUCAU) and its neighboring half site (UAAU)(Fig. 4A, TGE-wt). GLD-1 bound this wild-type TGE sequence and a variation of it (TGE-m2) with approximate Kd ~104 nM, defining a short sequence for GLD-1 high affinity binding (Fig. 4B). These data are consistent with previous competition experiments that defined the GLD-1 Kd ~10 nM that defined the hexanucleotide as (UACU(C/A)A) [37]. The nucleotide substitution of the half-site (UAAU to GAGU) abolished RNA binding (Fig. 4B, TGE-m1), consistent with the need for a half-site in addition to the hexanucleotide. Similar binding experiments were performed with QKI and we observed that TGE-m2 is essentially a QRE bound with high affinity, whereas the wild-type TGE bound with a moderate affinity of approximately 300 nM (Fig. 4C). The TGE-m1 was not bound by QKI (Fig. 4C). In summary, these data identify the GLD-1 RNA binding motif as bipartite as observed with SLM-2, QKI, and Sam68.


The STAR RNA binding proteins GLD-1, QKI, SAM68 and SLM-2 bind bipartite RNA motifs.

Galarneau A, Richard S - BMC Mol. Biol. (2009)

GLD-1 binding to the tra2/gli element analysis. (A) RNA species tested in (B) and (C) are shown. The black bars denote sequences that are unaltered between the wild-type and the mutant versions. EMSAs of the tra2/Gli element with increasing concentrations of GLD-1 (B) and QKI (C) (by a factor of 2 from 2 nM) or with buffer alone. Migration patterns of unbound RNAs (free probe) and protein/bound RNAs (GLD-1/RNA or QKI/RNA complexes) are indicated on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2697983&req=5

Figure 4: GLD-1 binding to the tra2/gli element analysis. (A) RNA species tested in (B) and (C) are shown. The black bars denote sequences that are unaltered between the wild-type and the mutant versions. EMSAs of the tra2/Gli element with increasing concentrations of GLD-1 (B) and QKI (C) (by a factor of 2 from 2 nM) or with buffer alone. Migration patterns of unbound RNAs (free probe) and protein/bound RNAs (GLD-1/RNA or QKI/RNA complexes) are indicated on the left.
Mentions: A high affinity RNA binding site has been defined for C. elegans GLD-1 that consists of a hexanucleotide (NACU(C/A)A) [37]. To examine whether the GLD-1 hexanucleotide sequence also requires a similar half-site, we performed EMSA assays with a segment of the tra2 and gli repeated element (TGE) containing the hexanucleotide (UACUCAU) and its neighboring half site (UAAU)(Fig. 4A, TGE-wt). GLD-1 bound this wild-type TGE sequence and a variation of it (TGE-m2) with approximate Kd ~104 nM, defining a short sequence for GLD-1 high affinity binding (Fig. 4B). These data are consistent with previous competition experiments that defined the GLD-1 Kd ~10 nM that defined the hexanucleotide as (UACU(C/A)A) [37]. The nucleotide substitution of the half-site (UAAU to GAGU) abolished RNA binding (Fig. 4B, TGE-m1), consistent with the need for a half-site in addition to the hexanucleotide. Similar binding experiments were performed with QKI and we observed that TGE-m2 is essentially a QRE bound with high affinity, whereas the wild-type TGE bound with a moderate affinity of approximately 300 nM (Fig. 4C). The TGE-m1 was not bound by QKI (Fig. 4C). In summary, these data identify the GLD-1 RNA binding motif as bipartite as observed with SLM-2, QKI, and Sam68.

Bottom Line: The GLD-1 binding site is defined as a hexanucleotide sequence (NACUCA) that is found in many, but not all, physiological GLD-1 mRNA targets.We also confirmed that GLD-1 also binds a bipartite RNA sequence in vitro with a short RNA sequence from its tra-2 physiological mRNA target.This information should help identify binding sites within physiological RNA targets.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bloomfield Center for Research on Aging, Lady Davis Institute for Medical Research, Sir Mortimer B, Davis Jewish General Hospital, and Department of Oncology, McGill University, Montréal, Québec, Canada. andre.galarneau@spcorp.com

ABSTRACT

Background: SAM68, SAM68-like mammalian protein 1 (SLM-1) and 2 (SLM-2) are members of the K homology (KH) and STAR (signal transduction activator of RNA metabolism) protein family. The function of these RNA binding proteins has been difficult to elucidate mainly because of lack of genetic data providing insights about their physiological RNA targets. In comparison, genetic studies in mice and C. elegans have provided evidence as to the physiological mRNA targets of QUAKING and GLD-1 proteins, two other members of the STAR protein family. The GLD-1 binding site is defined as a hexanucleotide sequence (NACUCA) that is found in many, but not all, physiological GLD-1 mRNA targets. Previously by using Systematic Evolution of Ligands by EXponential enrichment (SELEX), we defined the QUAKING binding site as a hexanucleotide sequence with an additional half-site (UAAY). This sequence was identified in QKI mRNA targets including the mRNAs for myelin basic proteins.

Results: Herein we report using SELEX the identification of the SLM-2 RNA binding site as direct U(U/A)AA repeats. The bipartite nature of the consensus sequence was essential for SLM-2 high affinity RNA binding. The identification of a bipartite mRNA binding site for QKI and now SLM-2 prompted us to determine whether SAM68 and GLD-1 also bind bipartite direct repeats. Indeed SAM68 bound the SLM-2 consensus and required both U(U/A)AA motifs. We also confirmed that GLD-1 also binds a bipartite RNA sequence in vitro with a short RNA sequence from its tra-2 physiological mRNA target.

Conclusion: These data demonstrate that the STAR proteins QKI, GLD-1, SAM68 and SLM-2 recognize RNA with direct repeats as bipartite motifs. This information should help identify binding sites within physiological RNA targets.

Show MeSH