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The reprogramming of alternative splicing networks during development is a hallmark of tissue maturation and identity. Alternative splicing of microexons (small, genomic regions ≤ 51 nucleotides) functionally regulate protein-protein interactions in the brain and is altered in several neuronal diseases. However, little is known about the regulation and function of alternatively spliced microexons in striated muscle. Here, we investigated alternative splicing of a microexon in the synaptosome-associated protein 23 (Snap23) encoded gene. We found that inclusion of this microexon is developmentally regulated and tissue-specific, as it occurs exclusively in adult heart and skeletal muscle. The alternative region is highly conserved in mammalian species and encodes an in-frame sequence of 11 amino acids. Furthermore, we showed that alternative splicing of this microexon is mis-regulated in mouse models of heart and skeletal muscle diseases. We identified the RNA-binding proteins (RBPs) quaking (QKI) and RNA binding fox-1 homolog 2 (RBFOX2) as the primary splicing regulators of the Snap23 microexon. We found that QKI and RBFOX2 bind downstream of the Snap23 microexon to promote its inclusion, and this regulation can be escaped when the weak splice donor is mutated to the consensus 5' splice site. Finally, we uncovered the interplay between QKI and muscleblind-like splicing regulator (MBNL) as an additional, but minor layer of Snap23 microexon splicing control. Our results are one of the few reports detailing microexon alternative splicing regulation during mammalian striated muscle development. 

The red-degraded [12.7]5Δ–X5Δ (0–0) band of gas-phase vanadium fluoride at 789 nm has been recorded by laser excitation spectroscopy and represents only the second reported rotational analysis of an electronic transition of VF. A hollow cathode discharge source was employed, with laser-induced fluorescence detected via the [12.7]5Δ–X5Δ (0–1) band. All five main (∆Ω = 0) subbands were identified, each containing only P and R branches, as expected in a parallel (∆Λ = 0) transition. The upper state displays the effects of strong local perturbations. Molecular constants describing the X5Δ (v = 0 and 1) levels and the [12.7]5Δ (v = 0) level were determined from least-squares fits using an effective Hamiltonian written in a Hund’s case (a) basis. The equilibrium rotational constant of the ground state was determined to be Be = 0.38324(89) cm−1, which yields a molecular bond length of Re = 1.7829(21) Å. 

ABSTRACT Spatial organization of pathway enzymes has emerged as a promising tool to address several challenges in metabolic engineering, such as flux imbalances and off-target product formation. Bacterial microcompartments (MCPs) are a spatial organization strategy used natively by many bacteria to encapsulate metabolic pathways that produce toxic, volatile intermediates. Several recent studies have focused on engineering MCPs to encapsulate heterologous pathways of interest, but how this engineering affects MCP assembly and function is poorly understood. In this study, we investigated the role of signal sequences, short domains that target proteins to the MCP core, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterized two novel Pdu signal sequences on the structural proteins PduM and PduB, which constitute the first report of metabolosome signal sequences on structural proteins rather than enzymes. We then explored the role of enzymatic and structural Pdu signal sequences on MCP assembly by deleting their encoding sequences from the genome alone and in combination. Deleting enzymatic signal sequences decreased the MCP formation, but this defect could be recovered in some cases by overexpressing genes encoding the knocked-out signal sequence fused to a heterologous protein. By contrast, deleting structural signal sequences caused similar defects to knocking out the genes encoding the full-length PduM and PduB proteins. Our results contribute to a growing understanding of how MCPs form and function in bacteria and provide strategies to mitigate assembly disruption when encapsulating heterologous pathways in MCPs.IMPORTANCESpatially organizing biosynthetic pathway enzymes is a promising strategy to increase pathway throughput and yield. Bacterial microcompartments (MCPs) are proteinaceous organelles that many bacteria natively use as a spatial organization strategy to encapsulate niche metabolic pathways, providing significant metabolic benefits. Encapsulating heterologous pathways of interest in MCPs could confer these benefits to industrially relevant pathways. Here, we investigate the role of signal sequences, short domains that target proteins for encapsulation in MCPs, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterize two novel signal sequences on structural proteins, constituting the first Pdu signal sequences found on structural proteins rather than enzymes, and perform knockout studies to compare the impacts of enzymatic and structural signal sequences on MCP assembly. Our results demonstrate that enzymatic and structural signal sequences play critical but distinct roles in Pdu MCP assembly and provide design rules for engineering MCPs while minimizing disruption to MCP assembly. 

Previously considered "housekeeping" genes, small nucleolar RNAs (snoRNAs) are increasingly understood to have wide-ranging functions in cancer, yet their role in metastasis has been less well studied. Here, we identify the snoRNA Snord67 as a regulator of lymph node (LN) metastasis in breast cancer. Snord67 expression is enriched in LN metastases in an immune-competent mouse model of female breast cancer. In an orthotopic breast cancer model, loss of Snord67 decreases LN metastasis. In a model of lymphatic metastasis, Snord67 loss decreases LN tumor growth and distant metastases. In breast cancer cell lines, Snord67 knockout results in loss of targeted 2'-O-methylation on U6 small nuclear RNA, as well as widespread changes in splicing. Together, these results demonstrate that Snord67 regulates splicing and promotes the growth of LN metastases and subsequent spread to distant metastases. SnoRNA-guided modifications of the spliceosome and regulation of splicing may represent a potentially targetable pathway in cancer. 

This mixed methods study investigates student learning outcomes from undergraduate STEM and non-STEM courses, employing farm-situated place-based experiential learning (PBEL) modules at a private liberal arts university in the Midwest. Given that these courses occurred during both COVID-19 and U.S. police brutality protests, this study critically interrogates the influence of this "dual pandemic" on student meaning-making. The study examines how student scores on environmental science literacy, civic-mindedness, sense of place, and scientific reasoning measures changed throughout the PBEL courses. With the exception of scientific reasoning, change in each measure was statistically significant (p<0.001). A stepwise linear regression determined whether any measures predicted civic-mindedness. Environmental science literacy and university place attachment were found to be predictive of civic-mindedness. Focus group data revealed how PBEL modules affected student learning outcomes Forand how the dual pandemic affected student civic-mindedness and place attachment. 

Abstract Surface deformation plays a key role in illuminating magma transport at active volcanoes, however, unambiguous separation of deep and shallow transport remains elusive. The Socorro Magma Body (SMB) lacks an upper crustal magma transport system, allowing us to link geodetic measurements with predictions of numerical models investigating rheologic heterogeneities and magma‐mush interaction in the mid‐/lower crust. New InSAR observations confirm that a pattern of central surface uplift surrounded by a region of subsidence (previously coined “sombrero” deformation) has persisted over >100 years at the SMB. Our models suggest this pattern may reflect the presence of a large (>100 km width), weaker‐than‐ambient, compliant region (CR) surrounding the mid‐crustal magma body. Interactions between a pressurizing (e.g., due to melt injection and/or volatile exsolution) sill‐like magma body and CR drive the sombrero pattern, depending on both viscoelastic relaxation and pressurization timescales, explaining its rare observation and transient nature. 

Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA- Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20–200 aM sensitivity. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.