Eukaryotic mRNA Processing Research Articles

Light-control of cap methylation and mRNA translation via genetic code expansion of Ecm1.

Gene expression is tightly regulated in all domains of life, with post-transcriptional regulation being more pronounced in higher eukaryotes. Optochemical and optogenetic approaches enable the actuation of many underlying processes by light, which is an excellent tool to exert spatio-temporal control. However, light-mediated control of eukaryotic mRNA processing and the respective enzymes has not been reported. We used genetic code expansion to install a photo-caged tyrosine (Y) in the active site of the cap methyltransferase Ecm1. This enzyme is responsible for guanine N7 methylation of the 5′ cap, which is required for translation. Substituting Y284 with the photocaged ortho-nitrobenzyl-tyrosine (ONBY) almost completely abrogated the methylation activity of Ecm1. Irradiation with light removed the ONB group, restoring the native tyrosine and Ecm1 activity, yielding up to 97% conversion of the minimal substrate GpppA within 60 min after activation. Using luciferase- and eGFP-mRNAs as reporters, we could show that light actuates translation by inducing activation of Ecm1 ONBY284 in a eukaryotic in vitro translation system.

Targeting Toxoplasma gondii CPSF3 as a new approach to control toxoplasmosis

Toxoplasma gondii is an important food and waterborne pathogen causing toxoplasmosis, a potentially severe disease in immunocompromised or congenitally infected humans. Available therapeutic agents are limited by suboptimal efficacy and frequent side effects that can lead to treatment discontinuation. Here we report that the benzoxaborole AN3661 had potent in vitro activity against T. gondii. Parasites selected to be resistant to AN3661 had mutations in TgCPSF3, which encodes a homologue of cleavage and polyadenylation specificity factor subunit 3 (CPSF‐73 or CPSF3), an endonuclease involved in mRNA processing in eukaryotes. Point mutations in TgCPSF3 introduced into wild‐type parasites using the CRISPR/Cas9 system recapitulated the resistance phenotype. Importantly, mice infected with T. gondii and treated orally with AN3661 did not develop any apparent illness, while untreated controls had lethal infections. Therefore, TgCPSF3 is a promising novel target of T. gondii that provides an opportunity for the development of anti‐parasitic drugs.

Ribonucleoprotein bodies are phased in.

Intracellular compartments are necessary for the regulation of many biochemical processes that ensure cell survival, growth and proliferation. Compartmentalisation is commonly achieved in organelles with defined lipid membranes, such as mitochondria, endoplasmic reticulum or the Golgi apparatus. While these organelles are responsible for many localised biochemical processes, recent evidence points to another class of compartments that lack membrane boundaries. The structure and content of these bodies depend on their function and subcellular localisation, but they mainly incorporate proteins and RNA. Examples of these ribonucleoprotein bodies (RNPBs) include eukaryotic mRNA processing bodies (P-bodies) and stress granules (SGs). While most of these structures have been widely studied for their capacity to bind, store and process mRNAs under different conditions, their biological functions and physical properties are poorly understood. Recent intriguing data suggest that liquid-liquid phase separation (LLPS) represents an important mechanism seeding the formation and defining the function of RNPBs. In this review, we discuss how LLPS is transforming our ideas about the biological functions of SGs and P-bodies and their link to diseases.

The union of transcription and mRNA processing: 20 years of coupling.

The union of transcription and mRNA processing: 20 years of coupling.

5′ to 3′ mRNA Decay Factors Colocalize with Ty1 Gag and Human APOBEC3G and Promote Ty1 Retrotransposition

The genomic RNA of retroviruses and retrovirus-like transposons must be sequestered from the cellular translational machinery so that it can be packaged into viral particles. Eukaryotic mRNA processing bodies (P bodies) play a central role in segregating cellular mRNAs from the translational machinery for storage or decay. In this work, we provide evidence that the RNA of the Saccharomyces cerevisiae Ty1 retrotransposon is packaged into virus-like particles (VLPs) in P bodies. Ty1 RNA is translationally repressed, and Ty1 Gag, the capsid and RNA binding protein, accumulates in discrete cytoplasmic foci, a subset of which localize to P bodies. Human APOBEC3G, a potent Ty1 restriction factor that is packaged into Ty1 VLPs via an interaction with Gag, also localizes to P bodies. The association of APOBEC3G with P bodies does not require Ty1 element expression, suggesting that P-body localization of APOBEC3G and Ty1 Gag precedes VLP assembly. Additionally, we report that two P-body-associated 5' to 3' mRNA decay pathways, deadenylation-dependent mRNA decay (DDD) and nonsense-mediated decay (NMD), stimulate Ty1 retrotransposition. The additive contributions of DDD and NMD explain the strong requirement for general 5' to 3' mRNA degradation factors Dcp1, Dcp2, and Xrn1 in Ty1 retromobility. 5' to 3' decay factors act at a posttranslational step in retrotransposition, and Ty1 RNA packaging into VLPs is abolished in the absence of the 5' to 3' exonuclease Xrn1. Together, the results suggest that VLPs assemble in P bodies and that 5' to 3' mRNA decay is essential for the packaging of Ty1 RNA in VLPs.

A simple answer for a splicing conundrum

A Simple Answer for a Splicing Conundrum. Douglas L. Black The medieval philosopher William of Occam admonished that it is best to minimize the postulated entities needed to explain a system. This concept of choosing the simplest explanation of unknown phenomena is now known as the Principle of Parsimony or Occam's Razor. However, in biology, where systems tend to evolve towards states of greater complexity, Occam's Razor can be a dangerous tool. Most often the known components of a biological process increase with study. It is not always safe to assume a limited number of factors are involved. Sometimes though, we do find a simple explanation of a puzzling phenomenon that requires no new unknowns to be added to the model. Such an event is described in this issue of PNAS, where three groups nicely explain a long-standing puzzle in eukaryotic mRNA processing [1]. Specifically, they demonstrate a plausible mechanism for how exons are always linked in order during the pre-mRNA splicing process, without a single exon being skipped. Eukaryotic mRNAs are transcribed as long precursor molecules containing intervening sequences, or introns separating the coding or otherwise functional portions of the transcript called exons (Figure 1A). To form a mature mRNA ready for translation, the cell must precisely excise the introns and ligate the exons together. This process of pre-mRNA splicing is carried out in the cell nucleus by a large macromolecular complex called the spliceosome [2]. Components of the spliceosome recognize special sequences at the intron ends called splice sites. The 5' splice site (at the 5' end of the intron) is initially bound by the U1 snRNP, and the 3' splice site is bound by the protein U2 Auxiliary Factor (U2AF) [3, 4]. These components interact to bring the two ends of the intron together, before going on to assemble the rest of the spliceosome that ultimately catalyzes the cleavage/ligation reactions. Early on, two questions of how splice sites are chosen for pairing during splicing were seen as particularly mysterious [5-7]. First, introns can be very long (>1000 nt.) and have within them many copies of the splice site consensus sequences. It was not understood how the correct sites at the intron ends were recognized, while the other cryptic sites were avoided. Second, pre-mRNAs can contain many exons that must

Nuclear export of hnRNP Hrp1p and nuclear export of hnRNP Npl3p are linked and influenced by the methylation state of Npl3p.

Eukaryotic mRNA processing and export are mediated by a series of complexes composed of heterogeneous nuclear ribonucleoproteins (hnRNPs). Many of these hnRNPs are methylated at arginine residues within their RGG domains. Although cellular arginine methylation is required for the efficient nuclear export of several hnRNPs, its role in this process is unknown. To address this question, we replaced the methylated RGG tripeptides of two hnRNPs, Npl3p and Hrp1p, with KGG. We found that these substitutions specifically abolish their methylation but have different effects on their nuclear export activity. Although the efficient export of Hrp1p requires cellular methyltransferase activity, the modification of Hrp1p itself is dispensable. In contrast, we found that Npl3 arginine methylation not only facilitates its own export but also is required for Hrp1p to efficiently exit the nucleus. Consistent with this observation, we found that Npl3p and Hrp1p exist in a ribonucleoprotein complex. We provide the first evidence that the arginine methylation of a particular protein directly affects its activity. Efficient export does not require methylation per se, but unmethylated arginine residues lead to retention of hnRNPs. Thus, arginine methylation serves to mask the Npl3p RGG domain for efficient ribonucleoprotein export.

Protein kinase A phosphorylation modulates transport of the polypyrimidine tract-binding protein.

The heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins play important roles in mRNA processing in eukaryotes, but little is known about how they are regulated by cellular signaling pathways. The polypyrimidine-tract binding protein (PTB, or hnRNP I) is an important regulator of alternative pre-mRNA splicing, of viral RNA translation, and of mRNA localization. Here we show that the nucleo-cytoplasmic transport of PTB is regulated by the 3',5'-cAMP-dependent protein kinase (PKA). PKA directly phosphorylates PTB on conserved Ser-16, and PKA activation in PC12 cells induces Ser-16 phosphorylation. PTB carrying a Ser-16 to alanine mutation accumulates normally in the nucleus. However, export of this mutant protein from the nucleus is greatly reduced in heterokaryon shuttling assays. Conversely, hyperphosphorylation of PTB by coexpression with the catalytic subunit of PKA results in the accumulation of PTB in the cytoplasm. This accumulation is again specifically blocked by the S16A mutation. Similarly, in Xenopus oocytes, the phospho-Ser-16-PTB is restricted to the cytoplasm, whereas the non-Ser-16-phosphorylated PTB is nuclear. Thus, direct PKA phosphorylation of PTB at Ser-16 modulates the nucleo-cytoplasmic distribution of PTB. This phosphorylation likely plays a role in the cytoplasmic function of PTB.

Interactions between mRNA export commitment, 3'-end quality control, and nuclear degradation.

Several aspects of eukaryotic mRNA processing are linked to transcription. In Saccharomyces cerevisiae, overexpression of the mRNA export factor Sub2p suppresses the growth defect of hpr1 null cells, yet the protein Hpr1p and the associated THO protein complex are implicated in transcriptional elongation. Indeed, we find that a pool of heat shock HSP104 transcripts are 3'-end truncated in THO complex mutant as well as sub2 mutant backgrounds. Surprisingly, however, this defect can be suppressed by deletion of the 3'-5' exonuclease Rrp6p. This indicates that incomplete RNAs result from nuclear degradation rather than from a failure to efficiently elongate transcription. RNAs that are not degraded are retained at the transcription site in a Rrp6p-dependent manner. Interestingly, the addition of a RRP6 deletion to sub2 or to THO complex mutants shows a strong synthetic growth phenotype, suggesting that the failure to retain and/or degrade defective mRNAs is deleterious. mRNAs produced in the 3'-end processing mutants rna14-3 and rna15-2, as well as an RNA harboring a 3' end generated by a self-cleaving hammerhead ribozyme, are also retained in Rrp6p-dependent transcription site foci. Taken together, our results show that several classes of defective RNPs are subject to a quality control step that impedes release from transcription site foci and suggest that suboptimal messenger ribonucleoprotein assembly leads to RNA degradation by Rrp6p.

Eukaryotic mRNA Processing. Adrian R. Krainer

Previous articleNext article No AccessNew Biological BooksEukaryotic mRNA Processing. Adrian R. Krainer Stephen A. LiebhaberStephen A. Liebhaber Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Quarterly Review of Biology Volume 74, Number 2Jun., 1999 Published in association with Stony Brook University Article DOIhttps://doi.org/10.1086/393085 Copyright 1999 The University of ChicagoPDF download Crossref reports no articles citing this article.

20 years of making messenger RNA: Eukaryotic mRNA Processing, Cold Spring Harbor Laboratory, NY, USA, 20–24 August 1997.

20 years of making messenger RNA: Eukaryotic mRNA Processing, Cold Spring Harbor Laboratory, NY, USA, 20–24 August 1997.

The HIV-1 Rev protein: prototype of a novel class of eukaryotic post-transcriptional regulators

Complex retroviruses, including Human Immunodeficiency Virus Type 1 (HIV-1), are characterized by the ordered temporal expression of the various viral gene products in infected cells. This effect is mediated by a novel class of RNA-sequence-specific regulatory proteins typified by the Rev trans-activator of HIV-1. Evidence suggests that Rev regulates HIV-1 gene expression by intervening in the normal pathway of eukaryotic mRNA processing and transport.

An analysis of the macronuclear actin genes of Oxytricha.

We have cloned and sequenced a 1.6-kb macronuclear molecule encoding actin from the hypotrichous ciliate Oxytricha nova. High-stringency Southern hybridization to native and digested macronuclear DNA shows that there is only one 1.6-kb actin-encoding molecule in O. nova. The 227-nucleotide 5' leader sequence contains AT-rich stretches punctuated by short GC regions. The AT-rich regions contain TATA-like sequences. However, other known eukaryote transcription regulatory sequences were not found. The 249-nucleotide 3' trailer sequence is also AT-rich and does not contain any obvious known eukaryotic mRNA processing signals. Sequence comparison with a closely related species, O. fallax, shows an 87% sequence similarity in the coding regions and an almost total lack of similarity in the noncoding regions of the molecules. However, a few small sequence similarities and motifs appear in the noncoding regions of the actin-encoding molecules of these two species. The actin-encoding molecule of O. nova could encode a polypeptide 374 amino acids long, the same size as some vertebrate cytoplasmic actins. Contrary to a previous report, we show that the actin-encoding molecule of O. fallax also codes for a polypeptide 374 amino acids long.