f RNA-binding proteins participate in a complex array of posttranscriptional controls essential to cell type specification and somatic development. Despite their detailed biochemical characterizations, the degree to which each RNA-binding protein impacts mammalian embryonic development remains incompletely defined, and the level of functional redundancy among subsets of these proteins remains open to question. The poly(C) binding proteins, PCBPs (␣CPs and hnRNP E proteins), are encoded by a highly conserved and broadly expressed gene family. The two major Pcbp isoforms, Pcbp2 and Pcbp1, are robustly expressed in a wide range of tissues and exert both nuclear and cytoplasmic controls over gene expression. Here, we report that Pcbp1-null embryos are rendered nonviable in the peri-implantation stage. In contrast, Pcbp2-null embryos undergo normal development until midgestation (12.5 to 13.5 days postcoitum), at which time they undergo a dramatic loss in viability associated with combined cardiovascular and hematopoietic abnormalities. Mice heterozygous for either Pcbp1 or Pcbp2 null alleles display a mild and nondisruptive defect in initial postpartum weight gain. These data reveal that Pcbp1 and Pcbp2 are individually essential for mouse embryonic development and have distinct impacts on embryonic viability and that Pcpb2 has a nonredundant in vivo role in hematopoiesis. These data further provide direct evidence that Pcbp1, a retrotransposed derivative of Pcpb2, has evolved an essential function(s) in the mammalian genome. P osttranscriptional control of gene expression plays a major role in eukaryotic cell type specification and organism development. RNA processing and mRNA expression are regulated in the nuclear and cytoplasmic compartments by a complex array of interactions among RNA-binding proteins, noncoding RNAs, and target transcripts (1, 2). Studies have documented the central importance of posttranscriptional controls in the programming of embryonic stem cell differentiation and somatic cell development (3-11). The critical role of posttranscriptional control of gene expression can be most clearly recognized in settings of transcriptional inactivity. For example, transcription is globally silenced in the differentiating erythroblast, and the terminal steps in erythrocyte formation are fully dependent on posttranscriptional controls over mRNA stability and translation (12-14). Studies of posttranscriptional controls in this and other models have relied heavily upon a variety of in vitro experimental platforms to define mechanisms and biochemical pathways. While highly informative, such studies do not address the in vivo relevance and nonredundant functions of RNA-binding proteins in physiologically intact environments.The PCBPs (also known as ␣CPs and heterogeneous ribonucleoprotein [hnRNP] E proteins) are a widely expressed and multifunctional family of RNA-binding proteins (15-19) that bind numerous erythroid and nonerythroid mRNAs (20,21). Studies focused on globin gene expression have revealed that t...

Background Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection. Results Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in “single pot” reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes. Conclusions LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.

Ribosome-profiling has uncovered pervasive translation in non-canonical open reading frames, however the biological significance of this phenomenon remains unclear. Using genetic variation from 71,702 human genomes, we assess patterns of selection in translated upstream open reading frames (uORFs) in 5’UTRs. We show that uORF variants introducing new stop codons, or strengthening existing stop codons, are under strong negative selection comparable to protein-coding missense variants. Using these variants, we map and validate gene-disease associations in two independent biobanks containing exome sequencing from 10,900 and 32,268 individuals, respectively, and elucidate their impact on protein expression in human cells. Our results suggest translation disrupting mechanisms relating uORF variation to reduced protein expression, and demonstrate that translation at uORFs is genetically constrained in 50% of human genes.

Formation of the mammalian hematopoietic system is under a complex set of developmental controls. Here, we report that mouse embryos lacking the KH domain poly(C) binding protein, Pcbp2, are selectively deficient in the definitive erythroid lineage. Compared to wild-type controls, transcript splicing analysis of the Pcbp2 embryonic liver reveals accentuated exclusion of an exon (exon 6) that encodes a highly conserved transcriptional control segment of the hematopoietic master regulator, Runx1. Embryos rendered homozygous for a Runx1 locus lacking this cassette exon (Runx1ΔE6) effectively phenocopy the loss of the definitive erythroid lineage in Pcbp2 embryos. These data support a model in which enhancement of Runx1 cassette exon 6 inclusion by Pcbp2 serves a critical role in development of hematopoietic progenitors and constitutes a critical step in the developmental pathway of the definitive erythropoietic lineage.

SARS-CoV-2 has caused a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but tests are expensive and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and Loop Mediated Isothermal Amplification (RT-LAMP) provides an alternative that uses alternative and often cheaper reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however a common artifact is nonspecific DNA amplification, complicating detection. Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions.

We have previously demonstrated that the two paralogous RNA binding protein, PCBP1 and PCBP2, are individually essential for mouse development: Pcbp1 -null embryos are peri-implantation lethal while Pcbp2 -null embryos lose viability at mid-gestation. Mid-gestation Pcbp2 −/− embryos revealed a complex phenotype that included loss of certain hematopoietic determinants. Whether PCBP2 directly contributes to erythropoietic differentiation and whether PCBP1 has a role in this process remained undetermined. Here we selectively inactivate the genes encoding these two RNA-binding proteins during differentiation of the erythroid lineage in the developing mouse embryo. Individual inactivation of either locus fails to impact viability or blood formation. However, combined inactivation of the two loci results in mid-gestational repression of erythroid/hematopoietic gene expression, loss of blood formation, and fetal demise. Orthogonal ex-vivo analyses of primary erythroid progenitors selectively depleted of these two RNA binding proteins revealed that they mediate a combination of overlapping and isoform-specific impacts on hematopoietic lineage transcriptome, impacting both mRNA representation and exon splicing. These data lead us to conclude that PCBP1 and PCBP2 mediate functions critical to differentiation of the erythroid lineage.

Ribosome-profiling has uncovered pervasive translation in 5'UTRs, however the biological significance of this phenomenon remains unclear. Using genetic variation from 71,702 human genomes, we assess patterns of selection in translated upstream open reading frames (uORFs) in 5'UTRs. We show that uORF variants introducing new stop codons, or strengthening existing stop codons, are under strong negative selection comparable to protein-coding missense variants. Using these variants, we map and validate new gene-disease associations in two independent biobanks containing exome sequencing from 10,900 and 32,268 individuals respectively, and demonstrate their impact on gene expression in human cells. Our results establish new mechanisms relating uORF variation to loss-of-function of downstream genes, and demonstrate that translated uORFs are genetically constrained regulatory elements in 40% of human genes.

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