The LARP1 homolog Slr1p controls the stability and expression of proto-5'TOP mRNAs in fission yeast.

Messenger RNAs (mRNAs) in higher eukaryotes that encode proteins important for the assembly of the translational apparatus (e.g., ribosomal proteins) often harbor a pyrimidine-rich motif at the extreme 5' end known as a 5' terminal oligopyrimidine (5'TOP) sequence. Members of the La-related protein 1 (LARP1) family control 5'TOP expression through a conserved DM15 motif, but the mechanism is not well understood. 5'TOP motifs have not been described in many lower organisms, and fission yeast harbors a LARP1 homolog that also lacks a DM15 motif. In this work, we show that the fission yeast LARP1 homolog, Slr1p, controls the translation and stability of mRNAs encoding proteins analogous to 5'TOP mRNAs in higher eukaryotes, which we thus refer to as proto-5'TOPs. Our data suggest that the LARP1 DM15 motif and the mRNA 5'TOP motif may be features that were scaffolded over a more fundamental mechanism of LARP1-associated control of gene expression.

Spatial positioning of preimplantation mouse embryo cells is regulated by mTORC1 and m7G-cap-dependent translation at the 8- to 16-cell transition.

Preimplantation mouse embryo development involves temporal-spatial specification and segregation of three blastocyst cell lineages: trophectoderm, primitive endoderm and epiblast. Spatial separation of the outer-trophectoderm lineage from the two other inner-cell-mass (ICM) lineages starts with the 8- to 16-cell transition and concludes at the 32-cell stages. Accordingly, the ICM is derived from primary and secondary contributed cells; with debated relative EPI versus PrE potencies. We report generation of primary but not secondary ICM populations is highly dependent on temporal activation of mammalian target of Rapamycin (mTOR) during 8-cell stage M-phase entry, mediated via regulation of the 7-methylguanosine-cap (m7G-cap)-binding initiation complex (EIF4F) and linked to translation of mRNAs containing 5' UTR terminal oligopyrimidine (TOP-) sequence motifs, as knockdown of identified TOP-like motif transcripts impairs generation of primary ICM founders. However, mTOR inhibition-induced ICM cell number deficits in early blastocysts can be compensated by the late blastocyst stage, after inhibitor withdrawal; compensation likely initiated at the 32-cell stage when supernumerary outer cells exhibit molecular characteristics of inner cells. These data identify a novel mechanism specifically governing initial spatial segregation of mouse embryo blastomeres, that is distinct from those directing subsequent inner cell formation, contributing to germane segregation of late blastocyst lineages.

RNA localization mechanisms transcend cell morphology.

RNA molecules are localized to specific subcellular regions through interactions between RNA regulatory elements and RNA binding proteins (RBPs). Generally, our knowledge of the mechanistic details behind the localization of a given RNA is restricted to a particular cell type. Here, we show that RNA/RBP interactions that regulate RNA localization in one cell type predictably regulate localization in other cell types with vastly different morphologies. To determine transcriptome-wide RNA spatial distributions across the apicobasal axis of human intestinal epithelial cells, we used our recently developed RNA proximity labeling technique, Halo-seq. We found that mRNAs encoding ribosomal proteins (RP mRNAs) were strongly localized to the basal pole of these cells. Using reporter transcripts and single-molecule RNA FISH, we found that pyrimidine-rich motifs in the 5' UTRs of RP mRNAs were sufficient to drive basal RNA localization. Interestingly, the same motifs were also sufficient to drive RNA localization to the neurites of mouse neuronal cells. In both cell types, the regulatory activity of this motif was dependent on it being in the 5' UTR of the transcript, was abolished upon perturbation of the RNA-binding protein LARP1, and was reduced upon inhibition of kinesin-1. To extend these findings, we compared subcellular RNAseq data from neuronal and epithelial cells. We found that the basal compartment of epithelial cells and the projections of neuronal cells were enriched for highly similar sets of RNAs, indicating that broadly similar mechanisms may be transporting RNAs to these morphologically distinct locations. These findings identify the first RNA element known to regulate RNA localization across the apicobasal axis of epithelial cells, establish LARP1 as an RNA localization regulator, and demonstrate that RNA localization mechanisms cut across cell morphologies.

The information required to build a specific protein is encoded into molecules of RNA which are often trafficked to precise locations in a cell. These journeys require a complex molecular machinery to be assembled and set in motion so that the RNA can be transported along dynamic 'roads' called microtubules. The details of this mechanism are known only for a handful of RNAs in a few cell types; for example, scientists have uncovered the signals presiding over the shuttling of certain RNAs to the axon, the long and thin projection that a neuron uses to communicate. Yet these RNAs are also present in cells that lack axons. Whether the molecular processes which preside over RNA movement apply across cell types has so far remained unclear. To investigate this question, Goering et al. tracked the location of RNA molecules in two types of polarized mouse cells: neurons which feature an axon, and 'epithelial' cells which line the intestine. The experiments revealed that the signals sending RNAs to the axons also directed the molecules towards the bottom pole of epithelial cells. In both cases, the RNAs travelled towards the extremity of the growing, "plus" end of the microtubules. Overall, this work suggests that RNA transport mechanisms should not be thought of as leading to a particular location in the cell; instead, they may be following more generalisable instructions. This knowledge could allow scientists to predict where a particular RNA will be sent across cell types based on data from one cell population. It could also aid the development of synthetic RNAs that target specific parts of the cell, offering greater control over their actions.

The molecular basis of mTORC1-regulated translation.

The mammalian target of rapamycin (mTOR) signaling pathway is a master regulator of cell growth throughout eukaryotes. The pathway senses nutrient and other growth signals, and then orchestrates the complex systems of anabolic and catabolic metabolism that underpin the growth process. A central target of mTOR signaling is the translation machinery. mTOR uses a multitude of translation factors to drive the bulk production of protein that growth requires, but also to direct a post-transcriptional program of growth-specific gene expression. This review will discuss current understanding of how mTOR controls these mechanisms and their functions in growth control.

Trans-splicing in metazoans: A link to translational control?

The trans-splicing of a spliced-leader RNA to a subset of mRNAs is a phenomenon that occurs in many species, including Caenorhabditis elegans, and yet the driving force for its evolution in disparate groups of animals remains unclear. Polycistronic mRNA resulting from the transcription of operons is resolved via trans-splicing, but operons comprise only a sub-set of trans-spliced genes. Using the marine chordate, Oikopleura dioica, we recently tested the hypothesis that metazoan operons accelerate recovery from growth arrest. We found no supporting evidence for this in O. dioica. Instead we found a striking relationship between trans-splicing and maternal mRNA in O. dioica, C. elegans and the ascidian, Ciona intestinalis. Furthermore, in O. dioica and C. elegans, we found evidence to suggest a role for mTOR signaling in the translational control of growth-related, trans-spliced maternal mRNAs. We propose that this may be a mechanism for adjusting egg number in response to nutrient levels in these species.

La-related Protein 1 (LARP1) Represses Terminal Oligopyrimidine (TOP) mRNA Translation Downstream of mTOR Complex 1 (mTORC1).

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5'TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1.