Abstract
The lysine methyltransferase (KMT) SETMAR is implicated in the response to and repair of DNA damage, but its molecular function is not clear. SETMAR has been associated with dimethylation of histone H3 lysine 36 (H3K36) at sites of DNA damage. However, SETMAR does not methylate H3K36 in vitro. This and the observation that SETMAR is not active on nucleosomes suggest that H3K36 methylation is not a physiologically relevant activity. To identify potential non-histone substrates, we utilized a strategy on the basis of quantitative proteomic analysis of methylated lysine. Our approach identified lysine 130 of the mRNA splicing factor snRNP70 as a SETMAR substrate in vitro, and we show that the enzyme primarily generates monomethylation at this position. Furthermore, we show that SETMAR methylates snRNP70 Lys-130 in cells. Because snRNP70 is a key early regulator of 5' splice site selection, our results suggest a model in which methylation of snRNP70 by SETMAR regulates constitutive and/or alternative splicing. In addition, the proteomic strategy described here is broadly applicable and is a promising route for large-scale mapping of KMT substrates.
Highlights
SETMAR is a lysine methyltransferase (KMT) that contributes to DNA repair, but its biochemical function is not well understood
Our proteomic approach identified two candidate SETMAR substrates, and we found that SETMAR methylates the splicing factor snRNP70 at lysine 130 both in vitro and in its cellular context
SETMAR Participates in Cellular Resistance to Topoisomerase II Inhibition—SETMAR has been shown to make a small contribution to cell growth in several cell lines, and it contributes to resistance to growth inhibition and cell death when cells are treated with inhibitors of Topo II [8, 9]
Summary
SETMAR is a lysine methyltransferase (KMT) that contributes to DNA repair, but its biochemical function is not well understood. Results: A novel proteomic strategy identifies splicing factor snRNP70 as a SETMAR substrate. Our approach identified lysine 130 of the mRNA splicing factor snRNP70 as a SETMAR substrate in vitro, and we show that the enzyme primarily generates monomethylation at this position. We recently reported a proteomic strategy to identify and measure lysine methylation by using the three malignant brain tumor domain repeats (3xMBT) of L3MBTL1 as a universal affinity reagent to capture modified proteins [10, 16]. SETMAR Methylates Splicing Factor snRNP70 that strategy into a generalized approach for identifying KMT substrates, and we use it to identify substrates of SETMAR. Our proteomic approach identified two candidate SETMAR substrates, and we found that SETMAR methylates the splicing factor snRNP70 ( called U1–70K) at lysine 130 both in vitro and in its cellular context
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