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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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STAR/GSG domain protein alignment. (A) Diagram representing the structural and functional region of the STAR/GSG domain containing proteins. (B) The STAR/GSG domain of mouse SLM-2, human SAM68, mouse QKI, C. elegans GLD-1 and human SF-1 were aligned using ClustalW. Secondary structure, beta sheets and alpha helices, are shown on top of the sequences and region NK/QUA1, the KH domain and region CK/QUA2 are shown beneath the sequences. The critical loop between helices alpha 1 and alpha 2 with the GXXG sequence is also shown. (a) Based on [6] the RNA bases UACUAAC that contact with the specific SF-1 residues are numbered as follow U1A2C3U4A5A6C7. (b) Arginine 160 makes contact with U4 and A6. (c) Valine 183 makes contact with A6 and C7.
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Figure 5: STAR/GSG domain protein alignment. (A) Diagram representing the structural and functional region of the STAR/GSG domain containing proteins. (B) The STAR/GSG domain of mouse SLM-2, human SAM68, mouse QKI, C. elegans GLD-1 and human SF-1 were aligned using ClustalW. Secondary structure, beta sheets and alpha helices, are shown on top of the sequences and region NK/QUA1, the KH domain and region CK/QUA2 are shown beneath the sequences. The critical loop between helices alpha 1 and alpha 2 with the GXXG sequence is also shown. (a) Based on [6] the RNA bases UACUAAC that contact with the specific SF-1 residues are numbered as follow U1A2C3U4A5A6C7. (b) Arginine 160 makes contact with U4 and A6. (c) Valine 183 makes contact with A6 and C7.

Mentions: The RNA binding domain of STAR/GSG proteins consist in a maxi-KH domain flanked by two conserved sequences (Fig. 5). The NK/QUA1 and CK/QUA2 region refer to the N- and C-terminal region, respectively, flanking the KH domain. Based on the structure of the KH domain of SF-1 associated with its binding RNA molecule U1A2C3U4A5A6C7, the CK region makes important contacts with the RNA. All STAR domain containing proteins have the most important GXXG sequence located in a loop between the two first alpha helices of the KH domain. This sequence of residues is absolutely conserved among the STAR domain proteins and makes the contact with the RNA especially with the bases U4A5A6C7. By looking closely at the residues in the CK region that make important association with the RNA bases, we find that two residues (asterix on Fig. 5) seems to confer the SLM-2/SAM68 specificity versus the QKI/GLD-1 specificity. The SLM-2/SAM68 residues are a threonine or a serine and a conserved glutamic acid while the QKI/GLD-1/SF1 residues consist in a conserved alanine and a conserved arginine. These residues make important contact with base A2 which specificity is lost in the Slm-2/Sam68 consensus binding sequence. In fact, SLM-2/Sam68 binding sequence resembles in all points to the QKI/GLD-1/SF1 core binding sequence but lacking U1A2C3 bases.


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

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

STAR/GSG domain protein alignment. (A) Diagram representing the structural and functional region of the STAR/GSG domain containing proteins. (B) The STAR/GSG domain of mouse SLM-2, human SAM68, mouse QKI, C. elegans GLD-1 and human SF-1 were aligned using ClustalW. Secondary structure, beta sheets and alpha helices, are shown on top of the sequences and region NK/QUA1, the KH domain and region CK/QUA2 are shown beneath the sequences. The critical loop between helices alpha 1 and alpha 2 with the GXXG sequence is also shown. (a) Based on [6] the RNA bases UACUAAC that contact with the specific SF-1 residues are numbered as follow U1A2C3U4A5A6C7. (b) Arginine 160 makes contact with U4 and A6. (c) Valine 183 makes contact with A6 and C7.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: STAR/GSG domain protein alignment. (A) Diagram representing the structural and functional region of the STAR/GSG domain containing proteins. (B) The STAR/GSG domain of mouse SLM-2, human SAM68, mouse QKI, C. elegans GLD-1 and human SF-1 were aligned using ClustalW. Secondary structure, beta sheets and alpha helices, are shown on top of the sequences and region NK/QUA1, the KH domain and region CK/QUA2 are shown beneath the sequences. The critical loop between helices alpha 1 and alpha 2 with the GXXG sequence is also shown. (a) Based on [6] the RNA bases UACUAAC that contact with the specific SF-1 residues are numbered as follow U1A2C3U4A5A6C7. (b) Arginine 160 makes contact with U4 and A6. (c) Valine 183 makes contact with A6 and C7.
Mentions: The RNA binding domain of STAR/GSG proteins consist in a maxi-KH domain flanked by two conserved sequences (Fig. 5). The NK/QUA1 and CK/QUA2 region refer to the N- and C-terminal region, respectively, flanking the KH domain. Based on the structure of the KH domain of SF-1 associated with its binding RNA molecule U1A2C3U4A5A6C7, the CK region makes important contacts with the RNA. All STAR domain containing proteins have the most important GXXG sequence located in a loop between the two first alpha helices of the KH domain. This sequence of residues is absolutely conserved among the STAR domain proteins and makes the contact with the RNA especially with the bases U4A5A6C7. By looking closely at the residues in the CK region that make important association with the RNA bases, we find that two residues (asterix on Fig. 5) seems to confer the SLM-2/SAM68 specificity versus the QKI/GLD-1 specificity. The SLM-2/SAM68 residues are a threonine or a serine and a conserved glutamic acid while the QKI/GLD-1/SF1 residues consist in a conserved alanine and a conserved arginine. These residues make important contact with base A2 which specificity is lost in the Slm-2/Sam68 consensus binding sequence. In fact, SLM-2/Sam68 binding sequence resembles in all points to the QKI/GLD-1/SF1 core binding sequence but lacking U1A2C3 bases.

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