Maximilian Wei-Lin Popp

Organizing Principles of Mammalian Nonsense-Mediated mRNA Decay

Cells use messenger RNAs (mRNAs) to ensure the accurate dissemination of genetic information encoded by DNA. Given that mRNAs largely direct the synthesis of a critical effector of cellular phenotype, i.e., proteins, tight regulation of both the quality and quantity of mRNA is a prerequisite for effective cellular homeostasis. Here, we review nonsense-mediated mRNA decay (NMD), which is the best-characterized posttranscriptional quality control mechanism that cells have evolved in their cytoplasm to ensure transcriptome fidelity. We use protein quality control as a conceptual framework to organize what is known about NMD, highlighting overarching similarities between these two polymer quality control pathways, where the protein quality control and NMD pathways intersect, and how protein quality control can suggest new avenues for research into mRNA quality control.

Leveraging Rules of Nonsense-Mediated mRNA Decay for Genome Engineering and Personalized Medicine

Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA quality control and regulatory process that plays direct roles in human health and disease. In this Minireview, we discuss how understanding the molecular events that trigger NMD can facilitate strategic targeting of genes via CRISPR/Cas9 technologies and also inform disease diagnostics and treatments.

The Dharma of Nonsense-Mediated mRNA Decay in Mammalian Cells

Mammalian-cell messenger RNAs (mRNAs) are generated in the nucleus from precursor RNAs (pre-mRNAs, which often contain one or more introns) that are complexed with an array of incompletely inventoried proteins. During their biogenesis, pre-mRNAs and their derivative mRNAs are subject to extensive cis-modifications. These modifications promote the binding of distinct polypeptides that mediate a diverse array of functions needed for mRNA metabolism, including nuclear export, inspection by the nonsense-mediated mRNA decay (NMD) quality-control machinery, and synthesis of the encoded protein product. Ribonucleoprotein complex (RNP) remodeling through the loss and gain of protein constituents before and after pre-mRNA splicing, during mRNA export, and within the cytoplasm facilitates NMD, ensuring integrity of the transcriptome. Here we review the mRNP rearrangements that culminate in detection and elimination of faulty transcripts by mammalian-cell NMD.

Attenuation of nonsense-mediated mRNA decay facilitates the response to chemotherapeutics

Nonsense-mediated mRNA decay (NMD) limits the production of aberrant mRNAs containing a premature termination codon and also controls the levels of endogenous transcripts. Here we show that when human cells are treated with clinically used chemotherapeutic compounds, NMD activity declines partly as a result of the proteolytic production of a dominant-interfering form of the key NMD factor UPF1. Production of cleaved UPF1 functions to upregulate genes involved in the response to apoptotic stresses. The biological consequence is the promotion of cell death. Combined exposure of cells to a small molecule inhibitor of NMD, NMDI-1, and the chemotherapeutic doxorubicin leads to enhanced cell death, while inhibiting UPF1 cleavage protects cells from doxorubicin challenge. We propose a model to explain why the expression levels of genes producing mRNAs of diverse structure that encode proteins of diverse function are under the purview of NMD.

Biochemical analysis of long non-coding RNA-containing ribonucleoprotein complexes

Long non-coding RNAs (lncRNAs), once relegated to junk products of the genome, are becoming better appreciated for the myriad functions they play in cellular processes. It is clear that for most of the cases studied, lncRNAs carry out their functions at least in part through interactions with proteins. Here we present two complementary biochemical methods for the analysis of lncRNA-containing ribonucleoprotein complexes, hereafter referred to as RNPs. The first strategy offers users the ability to purify RNPs based on a protein component and to analyze the spectrum of lncRNAs, other proteins, and, if present, other types of RNAs that are bound to it. The second makes use of a bacteriophage MS2 binding-site affinity-handle grafted onto an lncRNA of interest to investigate the proteins and RNAs that co-purify with the tagged RNA.

Nonsense-mediated mRNA Decay and Cancer

Nonsense-mediated mRNA decay (NMD) is a conserved mRNA surveillance pathway that cells use to ensure the quality of transcripts and to fine-tune transcript abundance. The role of NMD in cancer development is complex. In some cases, tumors have exploited NMD to downregulate gene expression by apparently selecting for mutations causing destruction of key tumor-suppressor mRNAs. In other cases, tumors adjust NMD activity to adapt to their microenvironment. Understanding how particular tumors exploit NMD for their benefit may augment the development of new therapeutic interventions.

Site-specific labeling of proteins via sortase: protocols for the molecular biologist.

Creation of site-specifically labeled protein bioconjugates is an important tool for the molecular biologist and cell biologist. Chemical labeling methods, while versatile with respect to the types of moieties that can be attached, suffer from lack of specificity, often targeting multiple positions within a protein. Here we describe protocols for the chemoenzymatic labeling of proteins at the C-terminus using the bacterial transpeptidase, sortase A. We detail a protocol for the purification of an improved pentamutant variant of the Staphylococcus aureus enzyme (SrtA 5(o)) that exhibits vastly improved kinetics relative to the wild-type enzyme. Importantly, a protocol for the construction of peptide probes compatible with sortase labeling using techniques that can be adapted to any cellular/molecular biology lab with no existing infrastructure for synthetic chemistry is described. Finally, we provide an example of how to optimize the labeling reaction using the improved SrtA 5(o) variant.

A Haploid Genetic Screening Method for Proteins Influencing Mammalian Nonsense-Mediated mRNA Decay Activity

Despite a long appreciation for the role of nonsense-mediated mRNA decay (NMD) in the destruction of faulty, disease-causing mRNAs, as well as its role in the maintenance of normal, endogenous transcript abundance, systematic unbiased methods for uncovering modifiers of NMD activity in mammalian cells remain scant. Here we present and validate a haploid genetic screening method for identifying proteins and processes that stimulate NMD activity involving a 3′-untranslated region exon-junction complex. This reporterbased screening method can be adapted for interrogating other pathways whose output can be measured by the intracellular production of fluorescent proteins.

A TRICK'n way to see the pioneer round of translation

Watching where and when individual messenger RNAs direct protein synthesis in live cells [Also see Report by Halstead et al.] Because proteins are largely the ultimate effectors of genotype, controlling the process of protein synthesis in space and time can play a critical role in cell metabolism. The subcellular localization of messenger RNAs (mRNAs) and their ensuing translation into proteins are known to be exploited to regulate processes such as development, cell motility, and neurotransmission. However, a dearth of tools for observing where and when mRNA translation occurs with single-molecule resolution in live cells has limited analysis to primarily ensemble, often static, biochemical measurements. On page 1367 of this issue, Halstead et al. (1) have filled this need by developing a method for visualizing mRNA translation in both live cells and whole animals.