SynLight: a bicistronic strategy for simultaneous active zone and cell labeling in the Drosophila nervous system.

At synapses, chemical neurotransmission mediates the exchange of information between neurons, leading to complex movement, behaviors, and stimulus processing. The immense number and variety of neurons within the nervous system make discerning individual neuron populations difficult, necessitating the development of advanced neuronal labeling techniques. In Drosophila, Bruchpilot-Short and mCD8-GFP, which label presynaptic active zones and neuronal membranes, respectively, have been widely used to study synapse development and organization. This labeling is often achieved via the expression of 2 independent constructs by a single binary expression system, but expression can weaken when multiple transgenes are expressed by a single driver. Recent work has sought to circumvent these drawbacks by developing methods that encode multiple proteins from a single transcript. Self-cleaving peptides, specifically 2A peptides, have emerged as effective sequences for accomplishing this task. We leveraged 2A ribosomal skipping peptides to engineer a construct that produces both Bruchpilot-Short-mStraw and mCD8-GFP from the same mRNA, which we named SynLight. Using SynLight, we visualized the putative synaptic active zones and membranes of multiple classes of olfactory, visual, and motor neurons and observed the correct separation of signal, confirming that both proteins are being generated separately. Furthermore, we demonstrate proof of principle by quantifying synaptic puncta number and neurite volume in olfactory neurons and finding no difference between the synapse densities of neurons expressing SynLight or neurons expressing both transgenes separately. At the neuromuscular junction, we determined that the synaptic puncta number labeled by SynLight was comparable to the endogenous puncta labeled by antibody staining. Overall, SynLight is a versatile tool for examining synapse density in any nervous system region of interest and allows new questions to be answered about synaptic development and organization.

SynLight: a dicistronic strategy for simultaneous active zone and cell labeling in the Drosophila nervous system.

At synapses, chemical neurotransmission mediates the exchange of information between neurons, leading to complex movement behaviors and stimulus processing. The immense number and variety of neurons within the nervous system makes discerning individual neuron populations difficult, necessitating the development of advanced neuronal labeling techniques. In Drosophila , Bruchpilot-Short and mCD8-GFP, which label presynaptic active zones and neuronal membranes, respectively, have been widely used to study synapse development and organization. This labeling is often achieved via expression of two independent constructs by a single binary expression system, but expression can weaken when multiple transgenes are expressed by a single driver. Ensuring adequate expression of each transgene is essential to enable more complex experiments; as such, work has sought to circumvent these drawbacks by developing methods that encode multiple proteins from a single transcript. Self-cleaving peptides, specifically 2A peptides, have emerged as effective sequences for accomplishing this task. We leveraged 2A ribosomal skipping peptides to engineer a construct that produces both Bruchpilot-Short and mCD8-GFP from the same mRNA, which we named SynLight. Using SynLight, we visualized the putative synaptic active zones and membranes of multiple classes of olfactory, visual, and motor neurons and observed correct separation of signal, confirming that both proteins are being generated separately. Furthermore, we demonstrate proof-of-principle by quantifying synaptic puncta number and neurite volume in olfactory neurons and finding no difference between the synapse densities of neurons expressing SynLight or neurons expressing both transgenes separately. At the neuromuscular junction, we determined that synaptic puncta number labeled by SynLight was comparable to endogenous puncta labeled by antibody staining. Overall, SynLight is a versatile tool for examining synapse density in any nervous system region of interest and allows new questions to be answered about synaptic development and organization.

Dynamic changes in RNA-protein interactions and RNA secondary structure in mammalian erythropoiesis.

Two features of eukaryotic RNA molecules that regulate their post-transcriptional fates are RNA secondary structure and RNA-binding protein (RBP) interaction sites. However, a comprehensive global overview of the dynamic nature of these sequence features during erythropoiesis has never been obtained. Here, we use our ribonuclease-mediated structure and RBP-binding site mapping approach to reveal the global landscape of RNA secondary structure and RBP-RNA interaction sites and the dynamics of these features during this important developmental process. We identify dynamic patterns of RNA secondary structure and RBP binding throughout the process and determine a set of corresponding protein-bound sequence motifs along with their dynamic structural and RBP-binding contexts. Finally, using these dynamically bound sequences, we identify a number of RBPs that have known and putative key functions in post-transcriptional regulation during mammalian erythropoiesis. In total, this global analysis reveals new post-transcriptional regulators of mammalian blood cell development.

RNA-Binding Proteins PCBP1 and PCBP2 Are Critical Determinants of Murine Erythropoiesis.

We previously demonstrated that the two paralogous RNA-binding proteins PCBP1 and PCBP2 are individually essential for mouse development: Pcbp1-null embryos are peri-implantation lethal, while Pcbp2-null embryos lose viability at midgestation. Midgestation 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 inactivated the genes encoding these two RNA-binding proteins during differentiation of the erythroid lineage in the developing mouse embryo. Individual inactivation of either locus failed to impact viability or blood formation. However, combined inactivation of the two loci resulted in midgestational 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.

A cytosine-rich splice regulatory determinant enforces functional processing of the human α-globin gene transcript.

The establishment of efficient and stable splicing patterns in terminally differentiated cells is critical to maintenance of specific functions throughout the lifespan of an organism. The human α-globin (hα-globin) gene contains 3 exons separated by 2 short introns. Naturally occurring α-thalassemia mutations that trigger aberrant splicing have revealed the presence of cryptic splice sites within the hα-globin gene transcript. How cognate (functional) splice sites are selectively used in lieu of these cryptic sites has remained unexplored. Here we demonstrate that the preferential selection of a cognate splice donor essential to functional splicing of the hα-globin transcript is dependent on the actions of an intronic cytosine (C)-rich splice regulatory determinant and its interacting polyC-binding proteins. Inactivation of this determinant by mutation of the C-rich element or by depletion of polyC-binding proteins triggers a dramatic shift in splice donor activity to an upstream, out-of-frame, cryptic donor. The essential role of the C-rich element in hα-globin gene expression is supported by its coevolution with the cryptic donor site in primate species. These data lead us to conclude that an intronic C-rich determinant enforces functional splicing of the hα-globin transcript, thus acting as an obligate determinant of hα-globin gene expression.

PolyC-binding proteins enhance expression of the CDK2 cell cycle regulatory protein via alternative splicing.

The PolyC binding proteins (PCBPs) impact alternative splicing of a subset of mammalian genes that are enriched in basic cellular functions. Here, we focus our analysis on PCBP-controlled cassette exon-splicing within the cell cycle control regulator cyclin-dependent kinase-2 (CDK2) transcript. We demonstrate that PCBP binding to a C-rich polypyrimidine tract (PPT) preceding exon 5 of the CDK2 transcript enhances cassette exon inclusion. This splice enhancement is U2AF65-independent and predominantly reflects actions of the PCBP1 isoform. Remarkably, PCBPs' control of CDK2 ex5 splicing has evolved subsequent to mammalian divergence via conversion of constitutive exon 5 inclusion in the mouse CDK2 transcript to PCBP-responsive exon 5 alternative splicing in humans. Importantly, exclusion of exon 5 from the hCDK2 transcript dramatically represses the expression of CDK2 protein with a corresponding perturbation in cell cycle kinetics. These data highlight a recently evolved post-transcriptional pathway in primate species with the potential to modulate cell cycle control.