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.

We have inserted the sequence 5'-AAG-GAGGU-3', which is complementary to the 3' terminus of Escherichia coli 16S rRNA, in a reading frame and analyzed its effect on the accuracy and overall rate of translation in vivo. Translation over the sequence yields a 50% ribosomal frameshift if the reading phase is A-AGG-AGG-U. The other two possible frames do not give shifts. The introduction of a UAA stop codon before (UAA-AGG-AGG-U) but not after (A-AGG-AGG-UAA) the AGG codons abolishes the frameshift. The change in the reading phase occurs exclusively' to the + 1 direction. Efficient frameshifting is also induced by the sequence A-AGA-AGA-U. The arginine codons AGG and AGA are read by minor tRNA. Suppression of frameshifting takes place when a gene for minor tRNAArg is introduced on a multicopy plasmid. We suggest that frameshifting during translation of the A-AGG-AGG-U sequence is due to the erroneous decoding of the tandem AGG codons and arises by depletion of tRNAAra. The complementarity of tandem AGG codons to the 3' terminus of 16S rRNA is a coincidence and apparently not related to the shift. Replacing the AGG-AGG sequence by the optimal arginine codons CGU-CGU does not increase the overall rate of translation.

Cancer is a disease in which cellular growth regulatory networks are disrupted. Lesions in well-characterized oncogenes and tumor suppressors often contribute to the dysregulation, but recent work has also uncovered the fundamental importance of enzymes that modulate the acetylation status of chromatin to the initiation or progression of cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are known to be involved in physiological cellular processes, such as transcription, cell cycle progression, gene silencing, differentiation, DNA replication, and genotoxic responses, but they are also increasingly being implicated in tumorigenesis. Butyrate is a short-chain fatty acid (SCFA) that acts as a HDAC inhibitor and is being clinically evaluated as an anti-neoplastic therapeutic, primarily because of its ability to impose cell cycle arrest, differentiation, and/or apoptosis in many tumor cell types, and its favorable safety profile in humans. Additionally, HDAC inhibitors could be used in combination with certain established antitumor therapeutics, such as those that target transcription, to augment clinical efficacy, and/or reduce toxicity. The molecular pathways of butyrate and related next-generation synthetic SCFAs in mediating these effects have not been fully elucidated, but HDAC inhibition is associated with regulation of critical cell cycle regulators, such as cyclin D, p21(CIP1/WAF1), and p27(KIP1). It is anticipated that a better understanding of this critical intersection between SCFAs, HDACs, and cell cycle control will lead to the design of novel treatment strategies for neoplasias. This review will summarize some of the recent research in these arenas of HDAC-directed cancer therapy and discuss the potential application of these agents in synergy with current chemotherapeutics.

Arginine is coded for by CGN (N = G, A, U, C), AGA and AGG. In Escherichia coli there is little tRNA for AGA and AGG and the use of these codons is strongly avoided in virtually all genes. Recently, we demonstrated that the presence of tandem AGA or AGG codons in mRNA causes frameshifts with high frequency. Here, we show that phaseshifts can be suppressed when cells are transformed with the gene for tRNA(T4Arg) or E. coli tRNA(argU,Arg) demonstrating that such errors are the result of tRNA depletion. Bacteriophage T4 encoded tRNA(Arg) (anticodon UCU) corrects shifts at AGA-AGA but not at AGG-AGG, suggesting that this tRNA can only read AGA. Similarly, comparison of the translational efficiencies in an argU (Ts) mutant and in its isogenic wild type parent indicates that argU tRNA (anticodon UCU) reads AGA but not AGG. An argU (Ts) mutant barely reads through AGA-AGA at 42 degrees C but translation of AGG-AGG is hardly, if at all, affected. Overexpression of argU+ relaxes the codon specificity. The thermosensitive mutant in argU, previously called dnaY because it is defective in DNA replication, can be complemented for growth by the gene for tRNA(T4Arg). This implies that the sole function of the argU gene product is to sustain protein synthesis and that its role in replication is probably indirect.

It was previously shown that mouse Pus1p (mPus1p), a pseudouridine synthase (PUS) known to modify certain transfer RNAs (tRNAs), can also bind with nuclear receptors (NRs) and function as a coactivator through pseudouridylation and likely activation of an RNA coactivator called steroid receptor RNA activator (SRA). Use of cell extract devoid of human Pus1p activity derived from patients with mitochondrial myopathy and sideroblastic anemia, however, still showed SRA-modifying activity suggesting that other PUS(s) can also target this coactivator. Here, we show that related mPus3p, which has a different tRNA specificity than mPus1p, also serves as a NR coactivator. However, in contrast to mPus1p, it does not stimulate sex steroid receptor activity, which is likely due to lack of binding to this class of NRs. As expected from their tRNA activities, in vitro pseudouridylation assays show that mPus3p and mPus1p modify different positions in SRA, although some may be commonly targeted. Interestingly, the order in which these enzymes modify SRA determines the total number of pseudouridines. mPus3p and SRA are mainly cytoplasmic; however, mPus3p and SRA are also localized in distinct nuclear subcompartments. Finally, we identified an in vivo modified position in SRA, U206, which is likely a common target for both mPus1p and mPus3p. When U206 is mutated to A, SRA becomes hyperpseudouridylated in vitro, and it acquires dominant-negative activity in vivo. Thus, Pus1p- and Pus3p-dependent pseudouridylation of SRA is a highly complex posttranscriptional mechanism that controls a coactivator-corepressor switch in SRA with major consequences for NR signaling.