Saccharomyces cerevisiae NRE1 and IRC24 Encode Paralogous Benzil Oxidoreductases.

Irc24p is a benzil oxidoreductase encoded on chromosome IX of Saccharomyces cerevisiae . We identified a putative paralog, Nre1p, encoded 284 bp downstream. Both proteins are small, cytoplasmic, and are 52% identical (70% similar). PANTHER and PFAM analysis of the amino acid sequences and rigid pairwise structure alignment predicted a conserved active site and Rossmann folds in both, implicating NADH or NADPH as likely cofactors. We purified hexahistidine-tagged Irc24p and Nre1p. Both proteins catalyze the reduction of the diketone benzil with similar kinetics and a preference for NADPH. This is the first demonstration of in vitro function for Nre1p.

Students authoring molecular case studies as a partial course-based undergraduate research experience (CURE) for lab instruction.

Understanding the relationship between protein structure and function is a core-learning goal in biochemistry. Students often struggle to visualize proteins as three-dimensional objects that interact with other molecules to affect its biochemical consequences. We describe here a partial course-based undergraduate research experiences that has students exploring protein structure and function hands-on while authoring a molecular case study intended for others to use.

Epstein-Barr viral microRNAs target caspase 3.

The Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that transforms B cells and causes several malignancies including Burkitt's lymphoma. EBV differentially expresses at least 49 mature microRNAs (miRNAs) during latency in various infected epithelial and B cells. Recent high-throughput studies and functional assays have begun to reveal the function of the EBV miRNAs suggesting roles in latency, cell cycle control, and apoptosis. In particular, the central executioner of apoptosis, Caspase 3 (CASP3), was proposed as a target of select EBV miRNAs. However, whether CASP3 is truly a target of EBV miRNAs, and if so, which specific miRNAs target CASP3 is still under debate. Based on previously published high-throughput biochemical data and a bioinformatic analysis of the entire CASP3 3'-UTR, we identified 12 EBV miRNAs that have one or more seed binding sites in the CASP3 3'-UTR. We individually tested all 12 miRNAs for repression of CASP3 in luciferase reporter assays, and nine showed statistically significant (P < 0.001) repression of a full-length CASP3 reporter. Further, three EBV miRNAs, including BART22, exhibited repression of endogenous CASP3 protein. These data confirm that CASP3 is a direct target of specific EBV BART miRNAs.

Alternative capture of noncoding RNAs or protein-coding genes by herpesviruses to alter host T cell function.

In marmoset T cells transformed by Herpesvirus saimiri (HVS), a viral U-rich noncoding (nc) RNA, HSUR 1, specifically mediates degradation of host microRNA-27 (miR-27). High-throughput sequencing of RNA after crosslinking immunoprecipitation (HITS-CLIP) identified mRNAs targeted by miR-27 as enriched in the T cell receptor (TCR) signaling pathway, including GRB2. Accordingly, transfection of miR-27 into human T cells attenuates TCR-induced activation of mitogen-activated protein kinases (MAPKs) and induction of CD69. MiR-27 also robustly regulates SEMA7A and IFN-γ, key modulators and effectors of T cell function. Knockdown or ectopic expression of HSUR 1 alters levels of these proteins in virally transformed cells. Two other T-lymphotropic γ-herpesviruses, AlHV-1 and OvHV-2, do not produce a noncoding RNA to downregulate miR-27 but instead encode homologs of miR-27 target genes. Thus, oncogenic γ-herpesviruses have evolved diverse strategies to converge on common targets in host T cells.

The "Observer Effect" in genome-wide surveys of protein-RNA interactions.

Recent technological advances have spurred genome-wide studies that afford insights into ribonucleoprotein biology and transcript regulation on an unprecedented scale. Here we review techniques currently used to obtain genome-wide profiles of RNA-protein interactions in living cells. We highlight recent studies of the mRNA-bound proteome and address pitfalls inherent in such investigations.

Association of Argonaute proteins and microRNAs can occur after cell lysis.

MicroRNA (miRNA) target identification is a challenging but important endeavor. Global analyses of the direct mRNA targets of miRNAs have relied heavily upon immunopurification techniques, wherein a core protein component of the miRNA-protein complex, Argonaute (Ago), is immunoprecipitated to isolate associated RNAs. This approach involves the assumption that the selected RNAs were bound to the Ago protein in vivo and that the methodology did not significantly perturb endogenous interactions or produce novel interaction artifacts. To test whether RNAs that coimmunoprecipitate with human Ago were bound in vivo or could associate post-cell lysis, we used an experimental approach that distinguishes between these two origins of interaction. We show that a transfected miRNA mimic, but not a plasmid-expressed miRNA, can interact with human Ago proteins post-lysis. Our results have important implications for the design of miRNP immunoprecipitation experiments.

EBV and human microRNAs co-target oncogenic and apoptotic viral and human genes during latency.

Epstein-Barr virus (EBV) controls gene expression to transform human B cells and maintain viral latency. High-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) identified mRNA targets of 44 EBV and 310 human microRNAs (miRNAs) in Jijoye (Latency III) EBV-transformed B cells. While 25% of total cellular miRNAs are viral, only three viral mRNAs, all latent transcripts, are targeted. Thus, miRNAs do not control the latent/lytic switch by targeting EBV lytic genes. Unexpectedly, 90% of the 1664 human 3'-untranslated regions targeted by the 12 most abundant EBV miRNAs are also targeted by human miRNAs via distinct binding sites. Half of these are targets of the oncogenic miR-17∼92 miRNA cluster and associated families, including mRNAs that regulate transcription, apoptosis, Wnt signalling, and the cell cycle. Reporter assays confirmed the functionality of several EBV and miR-17 family miRNA-binding sites in EBV latent membrane protein 1 (LMP1), EBV BHRF1, and host CAPRIN2 mRNAs. Our extensive list of EBV and human miRNA targets implicates miRNAs in the control of EBV latency and illuminates viral miRNA function in general.

Comprehensive analysis of Rhesus lymphocryptovirus microRNA expression.

Rhesus lymphocryptovirus (rLCV) and Epstein-Barr virus (EBV) are closely related gammaherpesviruses that infect and cause disease in rhesus monkeys and humans, respectively. Thus, rLCV is an important model system for EBV pathogenesis. Both rLCV and EBV express microRNAs (miRNAs), several conserved in sequence and genomic location. We have applied deep sequencing technology to obtain an inventory of rLCV miRNA expression in latently rLCV-infected monkey B cells. Our data confirm the presence of all previously identified mature rLCV miRNAs and have resulted in the discovery of 21 new mature miRNAs arising from previously identified precursor miRNAs (pre-miRNAs), as well as two novel pre-miRNAs (rL1-34 and rL1-35) that together generate four new mature miRNAs. Thus, the total number of rLCV-encoded pre-miRNAs is 35 and the total number of rLCV mature miRNAs is 68, the most of any virus examined. The exact 5' and 3' ends of all mature rLCV miRNAs were pinpointed, many showing marked sequence and length heterogeneity that could modulate function. We further demonstrate that rLCV mature miRNAs associate with Argonaute proteins in rLCV-infected B cells.

Analysis of p53-RNA interactions in cultured human cells.

Tumor suppressor p53 is a well-characterized transcription factor that binds DNA. More enigmatic are the RNA-binding properties of p53 and their physiological relevance. We used three sensitive co-immunoprecipitation methods in an attempt to detect RNAs that tightly associate with p53 in cultured human cells. Although recombinant p53 protein binds RNA in a sequence-nonspecific mode, we do not detect specific in vivo RNA binding by p53. These results suggest that RNA binding is prevented by post-translational p53 modifications. A ribonucleoprotein (not p53) is purified by multiple IgG monoclonal antibodies (including anti-p53 antibodies) from both p53 +/+ and p53 null cells. Caution is therefore required in interpreting RNA co-immunoprecipitation experiments. Though not formally excluded, these results do not support models in which p53 binds specific RNA partners in vivo.

p53 RNA interactions: new clues in an old mystery.

The p53 tumor suppressor protein is typically considered to be a sequence-specific DNA-binding transcription factor. However, reports over the last 15 years have described RNA binding by p53 in a variety of contexts, suggesting the possibility of new p53 functions. It is clear that p53-RNA interactions are mediated by a nucleic acid-binding domain of p53 independent of the sequence-specific core domain responsible for DNA recognition. Reports disagree on several aspects of the putative RNA interaction, including sequence specificity and biological relevance. Here we review the history and recent advances in the study of p53-RNA interactions. We argue that p53-RNA interactions are sequence nonspecific and depend on incomplete post-translational modification of the p53 C-terminal domain when the protein is expressed in heterologous systems. It is unknown what fraction of p53 protein exists in a state competent for RNA binding in vivo. Thus, potential physiological roles of p53-RNA interactions remain mysterious.

RNA-p53 interactions in vitro.

The tumor suppressor protein p53 is mutated in over half of human cancers. Despite 25 years of study, the complex regulation of this protein remains unclear. After serendipitously detecting RNA binding by p53 in the yeast three-hybrid system (Y3H), we are exploring the specificity and function of this interaction. Electrophoretic mobility shift assays show that full-length p53 binds equally to RNAs that are strongly distinguished in the Y3H. RNA binding blocks sequence-specific DNA binding by p53. The C-terminus of p53 is necessary and sufficient for strong RNA interaction in vitro. Mouse and human C-terminal p53 peptides have different affinities for RNA, and an acetylated human p53 C-terminal peptide does not bind RNA. Circular dichroism spectroscopy of p53 peptides shows that RNA binding does not induce a structural change in the p53 C-terminal peptide, and C-terminal peptides do not detectably affect the structure of RNA. These results demonstrate that p53 binds RNA with little sequence specificity, RNA binding has the potential to regulate DNA binding, and RNA-p53 interactions can be regulated by acetylation of the p53 C-terminus.

Recognition of RNA by the p53 tumor suppressor protein in the yeast three-hybrid system.

The p53 tumor suppressor protein is a homotetrameric transcription factor whose gene is mutated in nearly half of all human cancers. In an unrelated screen of RNA/protein interactions using the yeast three-hybrid system, we inadvertently detected p53 interactions with several different RNAs. A literature review revealed previous reports of both sequence-specific and -non-specific interactions between p53 and RNA. Using yeast three-hybrid selections to identify preferred RNA partners for p53, we failed to identify primary RNA sequences or obvious secondary structures required for p53 binding. The cationic p53 C-terminus was shown to be required for RNA binding in yeast. We show that while p53 strongly discriminates between certain RNAs in the yeast three-hybrid assay, the same RNAs are bound equally by p53 in vitro. We further show that the p53 RNA-binding preferences in yeast are mirrored almost exactly by a recombinant tetrameric form of the HIV-1 nucleocapsid (NC) protein thought to be a sequence-nonspecific RNA-binding protein. However, the possibility of specific RNA binding by p53 could not be ruled out because p53 and HIV-1 NC displayed certain differences in RNA-binding preference. We conclude that (1) p53 binds RNA in vivo, (2) RNA binding by p53 is largely sequence-nonspecific in the yeast nucleus, (3) some structure-specific RNA binding by p53 cannot be ruled out, and (4) caution is required when interpreting results of RNA screens in the yeast three-hybrid system because sequence-dependent differences in RNA folding and display can masquerade as sequence-dependent differences in protein recognition.