SummaryAccurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.

PTB (polypyrimidine-tract-binding protein) is a ubiquitous RNA-binding protein. It was originally identified as a protein with a role in splicing but it is now known to function in a large number of diverse cellular processes including polyadenylation, mRNA stability and translation initiation. Specificity of PTB function is achieved by a combination of changes in the cellular localization of this protein (its ability to shuttle from the nucleus to the cytoplasm is tightly controlled) and its interaction with additional proteins. These differences in location and trans-acting factor requirements account for the fact that PTB acts both as a suppressor of splicing and an activator of translation. In the latter case, the role of PTB in translation has been studied extensively and it appears that this protein is required for an alternative form of translation initiation that is mediated by a large RNA structural element termed an IRES (internal ribosome entry site) that allows the synthesis of picornaviral proteins and cellular proteins that function to control cell growth and cell death. In the present review, we discuss how PTB regulates these disparate processes.

Loss of the RNA binding protein FMRP causes Fragile X Syndrome (FXS), the most common cause of inherited intellectual disability, yet it is unknown how FMRP function varies across brain regions and cell types and how this contributes to disease pathophysiology. Here we use conditional tagging of FMRP and CLIP (FMRP cTag CLIP) to examine FMRP mRNA targets in hippocampal CA1 pyramidal neurons, a critical cell type for learning and memory relevant to FXS phenotypes. Integrating these data with analysis of ribosome-bound transcripts in these neurons revealed CA1-enriched binding of autism-relevant mRNAs, and CA1-specific regulation of transcripts encoding circadian proteins. This contrasted with different targets in cerebellar granule neurons, and was consistent with circadian defects in hippocampus-dependent memory in Fmr1 knockout mice. These findings demonstrate differential FMRP-dependent regulation of mRNAs across neuronal cell types that may contribute to phenotypes such as memory defects and sleep disturbance associated with FXS.

The RNA binding protein Larp1 was originally shown to be involved in spermatogenesis, embryogenesis and cell-cycle progression in Drosophila. Our data show that mammalian Larp1 is found in a complex with poly A binding protein and eukaryote initiation factor 4E and is associated with 60S and 80S ribosomal subunits. A reduction in Larp1 expression by siRNA inhibits global protein synthesis rates and results in mitotic arrest and delayed cell migration. Consistent with these data we show that Larp1 protein is present at the leading edge of migrating cells and interacts directly with cytoskeletal components. Taken together, these data suggest a role for Larp1 in facilitating the synthesis of proteins required for cellular remodelling and migration.