Figure 7. MALAT1 Modulates the Cellular Levels and Phosphorylation of SR Proteins.
(A) Immunoblot assays in control (scr) and MA-LAT1-depleted total cell extracts (AS1 and AS2) revealed increased levels of SRSF1 and SRSF2 in MALAT1-depleted cells. Relative fold increase in the levels of SR proteins in MALAT1-depleted cells: SRSF1, AS1 2.07 and AS2 3.13; SRSF2, AS1 3.15 and AS2 1.52. Note that the MALAT1-depleted cell extracts contained extra bands of SR proteins that show fast mobility.
(B and C) Control (lanes 1–4) and MALAT1 anti-sense oligonucleotides (B, AS1; and C, AS2) transfected (lanes 5–8) HeLa cells were extracted using 150 and 300 mM NaCl salt into soluble supernatant (s; lanes 1, 3, 5, and 7) and insoluble chromatin pellet (p; lanes 2, 4, 6, and 8) fractions and immunoblotted using SRSF1 antibody. Lane 9 (B) represents total cell extract treated with Antarctic phosphatase (+AP).
(D) Immunoblot analyses of control and MALAT1-depleted extracts (300 mM NaCl extracted) with 3C5 (Da), SRSF1 (Db), SRSF2 (Dc), U2AF-65 (Dd), B″-U2snRNP (De), SF3a60 (Df) antibodies. Orc2 and MEK were used as loading controls. (E) Hypothetical model depicting the role of MA-LAT1 in AS regulation. (Ea) In normal cells, MA-LAT1, by associating with SR proteins in nuclear speckles and in the nucleoplasm, regulates their recruitment to the pre-mRNA, thereby regulating AS. Here we have shown SAT1 pre-mRNA as an example that undergoes alternate exon exclusion in normal cells. However, in MALAT1-depleted cells (Eb), cellular levels of SR proteins are increased and are also present predominantly in the dephosphorylated form, resulting in changes in AS of pre-mRNA. In case of SAT1 pre-mRNA, MALAT1 depletion results in the inclusion of an alternative exon containing weak splice sites. See also Figure S6.