The pairing of 5′ and 3′ splice sites across an intron is a critical step in spliceosome formation and its regulation. Interactions that bring the two splice sites together during spliceosome assembly must occur with a high degree of specificity and fidelity to allow expression of functional mRNAs and make particular alternative splicing choices. Here, we report a new interaction between stem–loop 4 (SL4) of the U1 snRNA, which recognizes the 5′ splice site, and a component of the U2 small nuclear ribonucleoprotein particle (snRNP) complex, which assembles across the intron at the 3′ splice site. Using a U1 snRNP complementation assay, we found that SL4 is essential for splicing in vivo. The addition of free U1-SL4 to a splicing reaction in vitro inhibits splicing and blocks complex assembly prior to formation of the prespliceosomal A complex, indicating a requirement for a SL4 contact in spliceosome assembly. To characterize the interactions of this RNA structure, we used a combination of stable isotope labeling by amino acids in cell culture (SILAC), biotin/Neutravidin affinity pull-down, and mass spectrometry. We show that U1-SL4 interacts with the SF3A1 protein of the U2 snRNP. We found that this interaction between the U1 snRNA and SF3A1 occurs within prespliceosomal complexes assembled on the pre-mRNA. Thus, SL4 of the U1 snRNA is important for splicing, and its interaction with SF3A1 mediates contact between the 5′ and 3′ splice site complexes within the assembling spliceosome.

Erythropoiesis, the development of erythrocytes from hematopoietic stem cells, occurs through four phases: erythroid progenitor (EP) development, early erythropoiesis, terminal erythroid differentiation (TED), and maturation. According to the classical model that is based on immunophenotypic profiles of cell populations, each of these phases comprises multiple differentiation states that arise in a hierarchical manner. After segregation of lymphoid potential, erythroid priming begins during progenitor development and progresses through progenitor cell types that have multilineage potential. Complete separation of the erythroid lineage is achieved during early erythropoiesis with the formation of unipotent EPs: burst-forming unit-erythroid and colony-forming unit-erythroid. These erythroid-committed progenitors undergo TED and maturation, which involves expulsion of the nucleus and remodeling to form functional biconcave, hemoglobin-filled erythrocytes. In the last decade or so, many studies employing advanced techniques such as single-cell RNA-sequencing (scRNA-seq) as well as the conventional methods, including colony-forming cell assays and immunophenotyping, have revealed heterogeneity within the stem, progenitor, and erythroblast stages, and uncovered alternate paths for segregation of erythroid lineage potential. In this review, we provide an in-depth account of immunophenotypic profiles of all cell types within erythropoiesis, highlight studies that demonstrate heterogeneous erythroid stages, and describe deviations to the classical model of erythropoiesis. Overall, although scRNA-seq approaches have provided new insights, flow cytometry remains relevant and is the primary method for validation of novel immunophenotypes.

Oxygen is a critical noncellular component of the bone marrow microenvironment that plays an important role in the development of hematopoietic cell lineages. In this study, we investigated the impact of low oxygen (hypoxia) on ex vivo myeloerythroid differentiation of human cord blood-derived CD34+ hematopoietic stem and progenitor cells. We characterized the culture conditions to demonstrate that low oxygen inhibits cell proliferation and causes a metabolic shift in the stem and progenitor populations. We found that hypoxia promotes erythroid differentiation by supporting the development of progenitor populations. Hypoxia also increases the megakaryoerythroid potential of the common myeloid progenitors and the erythroid potential of megakaryoerythroid progenitors and significantly accelerates maturation of erythroid cells. Specifically, we determined that hypoxia promotes the loss of CD71 and the appearance of the erythroid markers CD235a and CD239. Further, evaluation of erythroid populations revealed a hypoxia-induced increase in proerythroblasts and in enucleation of CD235a+ cells. These results reveal the extensive role of hypoxia at multiple steps during erythroid development. Overall, our work establishes a valuable model for further investigations into the relationship between erythroid progenitors and/or erythroblast populations and their hypoxic microenvironment.

Significance: Pre-messenger RNA (pre-mRNA) splicing is a key regulatory step in gene expression. The splicing reaction is mediated by the spliceosome, a dynamic complex comprising five small nuclear ribonucleoproteins (snRNPs), which assembles onto each intron in multiple steps. We present detailed structural analysis and supporting functional data of an important protein–RNA interaction between human U1 and U2 snRNP. Our structure shows that an intrinsically disordered arginine-glycine (RGG/RG)–rich motif of a U2 snRNP subunit forms an RNA-sequence–specific connection with U1 snRNP. This study broadens the functional scope of unstructured RGG/RG-rich motifs in RNA binding proteins and provides a molecular basis of early steps of spliceosome assembly, which may help develop innovative therapeutic strategies against diseases originating from splicing defects.

Myeloid malignancies including myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia are characterized by abnormal proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). Reports on analysis of bone marrow samples from patients have revealed a high incidence of mutations in splicing factors in early stem and progenitor cell clones, but the mechanisms underlying transformation of HSPCs harboring these mutations remain unknown. Using ex vivo cultures of primary human CD34⁺ cells as a model, we find that mutations in splicing factors SRSF2 and U2AF1 exert distinct effects on proliferation and differentiation of HSPCs. SRSF2 mutations cause a dramatic inhibition of proliferation via a G2‐M phase arrest and induction of apoptosis. U2AF1 mutations, on the other hand, do not significantly affect proliferation. Mutations in both SRSF2 and U2AF1 cause abnormal differentiation by skewing granulo‐monocytic differentiation towards monocytes but elicit diverse effects on megakaryo‐erythroid differentiation. The SRSF2 mutations skew differentiation towards megakaryocytes whereas U2AF1 mutations cause an increase in the erythroid cell populations. These distinct functional consequences indicate that SRSF2 and U2AF1 mutations have cell context‐specific effects and that the generation of myeloid disease phenotype by mutations in the genes coding these two proteins likely involves different intracellular mechanisms. © AlphaMed Press 2018

During spliceosome assembly, interactions that bring the 5′ and 3′ ends of an intron in proximity are critical for the production of mature mRNA. Here, we report synergistic roles for the stem-loops 3 (SL3) and 4 (SL4) of the human U1 small nuclear RNA (snRNA) in maintaining the optimal U1 snRNP function, and formation of cross-intron contact with the U2 snRNP. We find that SL3 and SL4 bind distinct spliceosomal proteins and combining a U1 snRNA activity assay with siRNA-mediated knockdown, we demonstrate that SL3 and SL4 act through the RNA helicase UAP56 and the U2 protein SF3A1, respectively. In vitro analysis using UV crosslinking and splicing assays indicated that SL3 likely promotes the SL4-SF3A1 interaction leading to enhancement of A complex formation and pre-mRNA splicing. Overall, these results highlight the vital role of the distinct contacts of SL3 and SL4 in bridging the pre-mRNA bound U1 and U2 snRNPs during the early steps of human spliceosome assembly.