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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.

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.

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SLM-2 RNA ligands identified. (A) EMSAs of pooled RNAs identified in rounds 2, 4 and 6 using increasing concentrations of His-SLM-2. The protein/RNA complex was separated from the free probe on a native PAGE. The migration patterns of unbound RNAs (free probe) and protein bound RNAs (SLM-2/RNA complex) are indicated on the left. (B) The sequences of 8 unique RNAs bound to SLM-2 after six cycles of SELEX. Both identified motifs are aligned and black undermark. Illustrated, underneath the sequences is the probability matrix (graphic logo) based on all the 8 different sequences, depicting the relative frequency of each residue at each position within the selected motif.
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Figure 1: SLM-2 RNA ligands identified. (A) EMSAs of pooled RNAs identified in rounds 2, 4 and 6 using increasing concentrations of His-SLM-2. The protein/RNA complex was separated from the free probe on a native PAGE. The migration patterns of unbound RNAs (free probe) and protein bound RNAs (SLM-2/RNA complex) are indicated on the left. (B) The sequences of 8 unique RNAs bound to SLM-2 after six cycles of SELEX. Both identified motifs are aligned and black undermark. Illustrated, underneath the sequences is the probability matrix (graphic logo) based on all the 8 different sequences, depicting the relative frequency of each residue at each position within the selected motif.

Mentions: To identify the binding motif for the SLM-2 RNA binding protein, we performed SELEX to enrich for high affinity RNA ligands. Bacterial recombinant SLM-2 expressed as a histidine epitope tagged fusion protein was generated and purified for the assay. Synthetic RNAs were transcribed with the T7 RNA polymerase from DNA pools of 52-nucleotide random-mers estimated at a complexity of 1.0 × 1014 and we randomly sequenced 20 RNA molecules from the initial library and noted, as expected, that each sequence was unique [39]. The transcribed RNAs were generated in the presence of 32P-α-UTP such that the amount of specific SLM-2 bound RNAs could be measured after each round. After six cycles of selection, we observed an approximately 10% of binding of the initial input (not shown), demonstrating that we indeed had enriched specific sequences. To confirm the SELEX amplification of the SLM-2 specific RNA ligands, we performed a gel electromobility shift assays (EMSA) with purified pools of RNA transcripts isolated from rounds 2, 4 and 6. The RNAs were 32P-labelled and incubated with buffer or increasing concentration of His-SLM-2. The SLM-2/RNA complexes were observed as slow migrating complexes on native gel electrophoresis (Fig. 1A). More efficient RNA binding was observed in round 6 than rounds 2 and 4 (compare the free probe remaining from lanes 2 and 6 with lane 10). After round 6, the SLM-2 bound RNAs were converted into cDNAs, subcloned and sequenced. The sequence of 43 clones revealed that 11 clones were unique (Table 1). The clones were referred to as SLM-2 response element (SRE)-1 to 11. Class I RNAs contained a bipartite motif consisting of direct repeats of the sequence U(U/A)AA (Table 1). Our data show that the selected RNA aptamers contained a bipartite motif with direct repeats and the spacing between the repeats varied from 3 (SRE-3) to 25 (SRE-7) nucleotides (Table 1 and Fig. 1B). The 3 RNAs identified that did not contain the bipartite sequence (SRE-9, -10, -11) were grouped in Class II and since ~10% of RNAs from round 6 bound SLM-2, Class II RNAs are likely to represent non-binders. No apparent secondary structure was identified in the SREs using the prediction of RNA secondary structure program MFOLD (data not shown). Taken together, we have identified a bipartite motif consisting of direct repeats of the sequence U(U/A)AA as the SLM-2 RNA binding site.


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

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

SLM-2 RNA ligands identified. (A) EMSAs of pooled RNAs identified in rounds 2, 4 and 6 using increasing concentrations of His-SLM-2. The protein/RNA complex was separated from the free probe on a native PAGE. The migration patterns of unbound RNAs (free probe) and protein bound RNAs (SLM-2/RNA complex) are indicated on the left. (B) The sequences of 8 unique RNAs bound to SLM-2 after six cycles of SELEX. Both identified motifs are aligned and black undermark. Illustrated, underneath the sequences is the probability matrix (graphic logo) based on all the 8 different sequences, depicting the relative frequency of each residue at each position within the selected motif.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 1: SLM-2 RNA ligands identified. (A) EMSAs of pooled RNAs identified in rounds 2, 4 and 6 using increasing concentrations of His-SLM-2. The protein/RNA complex was separated from the free probe on a native PAGE. The migration patterns of unbound RNAs (free probe) and protein bound RNAs (SLM-2/RNA complex) are indicated on the left. (B) The sequences of 8 unique RNAs bound to SLM-2 after six cycles of SELEX. Both identified motifs are aligned and black undermark. Illustrated, underneath the sequences is the probability matrix (graphic logo) based on all the 8 different sequences, depicting the relative frequency of each residue at each position within the selected motif.
Mentions: To identify the binding motif for the SLM-2 RNA binding protein, we performed SELEX to enrich for high affinity RNA ligands. Bacterial recombinant SLM-2 expressed as a histidine epitope tagged fusion protein was generated and purified for the assay. Synthetic RNAs were transcribed with the T7 RNA polymerase from DNA pools of 52-nucleotide random-mers estimated at a complexity of 1.0 × 1014 and we randomly sequenced 20 RNA molecules from the initial library and noted, as expected, that each sequence was unique [39]. The transcribed RNAs were generated in the presence of 32P-α-UTP such that the amount of specific SLM-2 bound RNAs could be measured after each round. After six cycles of selection, we observed an approximately 10% of binding of the initial input (not shown), demonstrating that we indeed had enriched specific sequences. To confirm the SELEX amplification of the SLM-2 specific RNA ligands, we performed a gel electromobility shift assays (EMSA) with purified pools of RNA transcripts isolated from rounds 2, 4 and 6. The RNAs were 32P-labelled and incubated with buffer or increasing concentration of His-SLM-2. The SLM-2/RNA complexes were observed as slow migrating complexes on native gel electrophoresis (Fig. 1A). More efficient RNA binding was observed in round 6 than rounds 2 and 4 (compare the free probe remaining from lanes 2 and 6 with lane 10). After round 6, the SLM-2 bound RNAs were converted into cDNAs, subcloned and sequenced. The sequence of 43 clones revealed that 11 clones were unique (Table 1). The clones were referred to as SLM-2 response element (SRE)-1 to 11. Class I RNAs contained a bipartite motif consisting of direct repeats of the sequence U(U/A)AA (Table 1). Our data show that the selected RNA aptamers contained a bipartite motif with direct repeats and the spacing between the repeats varied from 3 (SRE-3) to 25 (SRE-7) nucleotides (Table 1 and Fig. 1B). The 3 RNAs identified that did not contain the bipartite sequence (SRE-9, -10, -11) were grouped in Class II and since ~10% of RNAs from round 6 bound SLM-2, Class II RNAs are likely to represent non-binders. No apparent secondary structure was identified in the SREs using the prediction of RNA secondary structure program MFOLD (data not shown). Taken together, we have identified a bipartite motif consisting of direct repeats of the sequence U(U/A)AA as the SLM-2 RNA binding site.

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