STARs in the CNS

Ehrmann, Ingrid; Fort, Philippe; Elliott, David J.

doi:10.1042/bst20160084

Cited by 10 publications

(14 citation statements)

References 58 publications

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“…The insertion was accompanied by a 420 kb deletion directly upstream of the insertion site (Figures 5A – D ). The 3′ end of the TYW3 transgene is 7 kb upstream of the initiation codon of the Khdrbs2 gene (Figures 5D,E ), which encodes an RNA binding regulator of alternative splicing also called Slm1 ( Ehrmann et al, 2016 ). We validated the insertion site with primers spanning the junction between the 3′ end of Chromosome 1 and the 5′ end of the transgene (Figure 5F ), as well as the deletion caused by the insertion using primers for a sequence within this putatively deleted region (Figure 5G ).…”

Section: Resultsmentioning

confidence: 99%

“…We observed no overt phenotype, consistent with the finding that even Slm1 null mutants are viable, fertile and outwardly normal ( Iijima et al, 2014 ; Traunmüller et al, 2014 ). Nonetheless, Khdrbs2 (Slm1) and its homologues, Khdrbs1 (Sam68) and Khdrbs3 (Slm2) are known to regulate alternative splicing of critical neuronal genes ( Ehrmann et al, 2016 ) so alterations in its activity could affect neuronal development or function. Likewise, Fat4 has been implicated in several developmental processes, with the null mutant being neonatally lethal ( Saburi et al, 2008 ).…”

Section: Discussionmentioning

confidence: 99%

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Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes

Laboulaye

Duan

Qiao

et al. 2018

Front. Mol. Neurosci.

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Transgenic mouse lines are routinely employed to label and manipulate distinct cell types. The transgene generally comprises cell-type specific regulatory elements linked to a cDNA encoding a reporter or other protein. However, off-target expression seemingly unrelated to the regulatory elements in the transgene is often observed, it is sometimes suspected to reflect influences related to the site of transgene integration in the genome. To test this hypothesis, we used a proximity ligation-based method, Targeted Locus Amplification (TLA), to map the insertion sites of three well-characterized transgenes that appeared to exhibit insertion site-dependent expression in retina. The nearest endogenous genes to transgenes HB9-GFP, Mito-P, and TYW3 are Cdh6, Fat4 and Khdrbs2, respectively. For two lines, we demonstrate that expression reflects that of the closest endogenous gene (Fat4 and Cdh6), even though the distance between transgene and endogenous gene is 550 and 680 kb, respectively. In all three lines, the transgenes decrease expression of the neighboring endogenous genes. In each case, the affected endogenous gene was expressed in at least some of the cell types that the transgenic line has been used to mark and study. These results provide insights into the effects of transgenes and endogenous genes on each other’s expression, demonstrate that mapping insertion site is valuable for interpreting results obtained with transgenic lines, and indicate that TLA is a reliable method for integration site discovery.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes

Laboulaye

Duan

Qiao

et al. 2018

Front. Mol. Neurosci.

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“…The 3' end of the TYW3 transgene is 7 kb upstream of the initiation codon of the Khdrbs2 gene (Fig. 5d, e), which encodes an RNA binding regulator of alternative splicing also called Slm1 (Ehrmann et al, 2016). We validated the insertion site with primers spanning the junction between the 3' end of Chromosome 1 and the 5' end of the transgene (Fig.…”

Section: The Tyw3 Transgene Is Inserted Near Khdrbs2 On Chromosomementioning

confidence: 99%

“…We observed no overt phenotype, consistent with the finding that even Slm1 null mutants are viable, fertile and outwardly normal (Iijima et al, 2014Traunmuller et al, 2014. Nonetheless, Khdrbs2 (Slm1) and its homologues, Khdrbs1 (Sam68) and Khdrbs3 (Slm2) are known to regulate alternative splicing of critical neuronal genes (Ehrmann, et. al, 2016) so alternations in its activity could affect neuronal development or function.…”

Section: Transgenes Affect Expression Of Neighboring Endogenous Genesmentioning

confidence: 99%

Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes

Laboulaye

Duan

Qiao

et al. 2018

Preprint

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Transgenic mouse lines are routinely employed to label and manipulate distinct cell types. The transgene generally comprises cell-type specific regulatory elements linked to a cDNA encoding a reporter or other proteins. However, off-target expression seemingly unrelated to the regulatory elements in the transgene is often observed, and sometimes suspected to reflect influences related to the site of transgene integration in the genome. To test this hypothesis, we used a proximity ligation-based method, Targeted Locus Amplification (TLA), to map the insertion sites of three well-characterized transgenes that appeared to exhibit insertion site-dependent expression in retina. The nearest endogenous genes to transgenes HB9-GFP, Mito-P, and TYW3 are Cdh6, Fat4 and Khdrbs2, respectively. For two lines, we demonstrate that expression reflects that of the closest endogenous gene (Fat4 and Cdh6), even though the distance between transgene and endogenous gene is 550 and 680 kb, respectively. In all three lines, the transgenes decrease expression of the neighboring endogenous genes. In each case, the affected endogenous gene was expressed in at least some of the cell types that the transgenic line has been used to mark and study. These results provide insights into the effects of transgenes and endogenous genes on each other's expression, demonstrate that mapping insertion site is valuable for interpreting results obtained with transgenic lines, and indicate that TLA is a reliable method for integration site discovery.

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“…SLM2 and Sam68 are 62% identical, and have similar modular organisations, including a STAR domain comprising a KH domain and flanking regions that mediate protein–protein and protein–RNA interactions, an RG (arginine/glycine)-rich region and C-termini enriched in tyrosine residues (14). Based on atomic level resolution from X-ray crystallography and nuclear magnetic resonance (15), SLM2 and Sam68 proteins bind RNA via their STAR domains, with largely overlapping but not identical RNA–protein contacts with the same U(A/U)AA target sequence (abbreviated UWAA) (15–17).…”

Section: Introductionmentioning

confidence: 99%

Binding site density enables paralog-specific activity of SLM2 and Sam68 proteins in Neurexin2 AS4 splicing control

et al. 2016

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SLM2 and Sam68 are splicing regulator paralogs that usually overlap in function, yet only SLM2 and not Sam68 controls the Neurexin2 AS4 exon important for brain function. Herein we find that SLM2 and Sam68 similarly bind to Neurexin2 pre-mRNA, both within the mouse cortex and in vitro. Protein domain-swap experiments identify a region including the STAR domain that differentiates SLM2 and Sam68 activity in splicing target selection, and confirm that this is not established via the variant amino acids involved in RNA contact. However, far fewer SLM2 and Sam68 RNA binding sites flank the Neurexin2 AS4 exon, compared with those flanking the Neurexin1 and Neurexin3 AS4 exons under joint control by both Sam68 and SLM2. Doubling binding site numbers switched paralog sensitivity, by placing the Neurexin2 AS4 exon under joint splicing control by both Sam68 and SLM2. Our data support a model where the density of shared RNA binding sites around a target exon, rather than different paralog-specific protein–RNA binding sites, controls functional target specificity between SLM2 and Sam68 on the Neurexin2 AS4 exon. Similar models might explain differential control by other splicing regulators within families of paralogs with indistinguishable RNA binding sites.

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STARs in the CNS

Cited by 10 publications

References 58 publications

Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes

Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes

Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes

Binding site density enables paralog-specific activity of SLM2 and Sam68 proteins in Neurexin2 AS4 splicing control

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