Regulation of Nuclear Receptor Activity by a Pseudouridine Synthase through Posttranscriptional Modification of Steroid Receptor RNA Activator

Abstract

Nuclear receptors (NRs) induce transcription through association with coactivator complexes. We identified a pseudouridine synthase (PUS), mPus1p, as a coactivator for retinoic acid receptor (mRAR)gamma and other NR-dependent transactivation. mPus1p is a member of the truA subfamily of PUSs, a class of enzymes that isomerize uridine to pseudouridine in noncoding RNAs, such as tRNA, to ensure proper folding and function. mPus1p binds the first zinc finger of mRARgamma and also associates with other NRs. Interestingly, mPus1p pseudouridylates coactivator Steroid Receptor RNA Activator (SRA), and when coexpressed, mPus1p and SRA cooperatively enhance mRARgamma-mediated transcription. mPus1p, mRARgamma, and SRA exist in a retinoid-independent, promoter bound complex in the nucleus although mPus1p is also expressed in the nucleolus, where it likely modifies tRNA. Finally, we show that mPus1p-coactivator function required SRA, mPus1p-associated mRARgamma binding, and PUS activities. mPus1p-dependent pseudouridylation of SRA represents an additional type of posttranscriptional modification of a NR-coactivator complex that is important for NR signaling.

Regulation of MITF stability by the USP13 deubiquitinase

“…The results in Figure 3A demonstrate a high efficiency of MITF polyubiquitination (compare lanes 1 and 3) under the applied reaction conditions. ubMITF was seen to be efficiently deubiquitinated by USP13 (compare lanes 3 and 4) without influencing the nonubiquitinated 35 S-MITF pattern (compare lanes 1 and 2), suggesting that USP13 triggers MITF deubiquitination.…”

“…Although our experiments suggest that USP13 stabilizes MITF and influences the level of ubMITF in cells, biochemical studies were carried out to more directly examine whether USP13 deubiquitinates MITF. Utilizing a 'TNT' in vitro coupled transcription/translation system, 35 S-labelled MITF and nonradioactive USP13 were produced in rabbit reticulocyte extracts (Promega). To make ubiquitinated MITF (ubMITF), polyubiquitin chain (UB 2-7 ; Boston Biochem) was conjugated to 35 S-MITF under the catalytic action of TIPP (Thermostable Inorganic Pyrophosphatase) and ATP 18 .…”

“…Utilizing a 'TNT' in vitro coupled transcription/translation system, 35 S-labelled MITF and nonradioactive USP13 were produced in rabbit reticulocyte extracts (Promega). To make ubiquitinated MITF (ubMITF), polyubiquitin chain (UB 2-7 ; Boston Biochem) was conjugated to 35 S-MITF under the catalytic action of TIPP (Thermostable Inorganic Pyrophosphatase) and ATP 18 . To study the deubiquitinating activity of USP13, 35 S-ubMITF was mixed with USP13 to perform a deubiquitination assay 18 .…”

“…To make ubiquitinated MITF (ubMITF), polyubiquitin chain (UB 2-7 ; Boston Biochem) was conjugated to 35 S-MITF under the catalytic action of TIPP (Thermostable Inorganic Pyrophosphatase) and ATP 18 . To study the deubiquitinating activity of USP13, 35 S-ubMITF was mixed with USP13 to perform a deubiquitination assay 18 . Equal quantities of 35 S-MITF from each reaction were loaded onto SDS-PAGE gels for analysis.…”

“…Since the E3 ligase involved in MITF ubiquitination is uncertain, we set up an experimental protocol in which melanoma cell lysate (to provide essential factors for MITF ubiquitination) was mixed with ATP, ubiquitin conjugating enzyme fractions (Boston Biochem) and 35 S-MITF. As the incubation proceeded, the amount of free 35 S-MITF decreased while the amount of ubMITF increased (Fig. 3b, lanes 6-8).…”