The intronic branch point sequence is under strong evolutionary constraint in the bovine and human genome

The branch point sequence is a cis-acting intronic motif required for mRNA splicing. Despite their functional importance, branch point sequences are not routinely annotated. Here we predict branch point sequences in 179,476 bovine introns and investigate their variability using a catalogue of 29.4 million variants detected in 266 cattle genomes. We localize the bovine branch point within a degenerate heptamer “nnyTrAy”. An adenine residue at position 6, that acts as branch point, and a thymine residue at position 4 of the heptamer are more strongly depleted for mutations than coding sequences suggesting extreme purifying selection. We provide evidence that mutations affecting these evolutionarily constrained residues lead to alternative splicing. We confirm evolutionary constraints on branch point sequences using a catalogue of 115 million SNPs established from 3,942 human genomes of the gnomAD database.

U2 snRNA structure is influenced by SF3A and SF3B proteins but not by SF3B inhibitors

U2 snRNP is an essential component of the spliceosome. It is responsible for branch point recognition in the spliceosome A-complex via base-pairing of U2 snRNA with an intron to form the branch helix. Small molecule inhibitors target the SF3B component of the U2 snRNP and interfere with A-complex formation during spliceosome assembly. We previously found that the first SF3B inhibited-complex is less stable than A-complex and hypothesized that SF3B inhibitors interfere with U2 snRNA secondary structure changes required to form the branch helix. Using RNA chemical modifiers, we probed U2 snRNA structure in A-complex and SF3B inhibited splicing complexes. The reactivity pattern for U2 snRNA in the SF3B inhibited-complex is indistinguishable from that of A-complex, suggesting that they have the same secondary structure conformation, including the branch helix. This observation suggests SF3B inhibited-complex instability does not stem from an alternate RNA conformation and instead points to the inhibitors interfering with protein component interactions that normally stabilize U2 snRNP’s association with an intron. In addition, we probed U2 snRNA in the free U2 snRNP in the presence of SF3B inhibitor and again saw no differences. However, increased protection of nucleotides upstream of Stem I in the absence of SF3A and SF3B proteins suggests a change of secondary structure at the very 5′ end of U2 snRNA. Chemical probing of synthetic U2 snRNA in the absence of proteins results in similar protections and predicts a previously uncharacterized extension of Stem I. Because this stem must be disrupted for SF3A and SF3B proteins to stably join the snRNP, the structure has the potential to influence snRNP assembly and recycling after spliceosome disassembly.

The Partitioning of Newly Assimilated Linoleic and α-Linolenic Acids Between Synthesis of Longer-Chain Polyunsaturated Fatty Acids and Hydroxyoctadecaenoic Acids Is a Putative Branch Point in T-Cell Essential Fatty Acid Metabolism

Longer-chain polyunsaturated fatty acids (LCPUFAs) ≥20 carbons long are required for leukocyte function. These can be obtained from the diet, but there is some evidence that leukocytes can convert essential fatty acids (EFAs) into LCPUFAs. We used stable isotope tracers to investigate LCPUFA biosynthesis and the effect of different EFA substrate ratios in human T lymphocytes. CD3+ T cells were incubated for up to 48 h with or without concanavalin A in media containing a 18:2n-6:18:3n-3 (EFA) ratio of either 5:1 or 8:1 and [13C]18:3n-3 plus [d5]18:2n-6. Mitogen stimulation increased the amounts of 16:1n-7, 18:1n-9, 18:2n-6, 20:3n-6, 20:4n-6, 18:3n-3, and 20:5n-3 in T cells. Expression of the activation marker CD69 preceded increased FADS2 and FADS1 mRNA expression and increased amounts of [d5]20:2n-6 and [13C]20:3n-3 at 48 h. In addition, 22-carbon n-6 or n-3 LCPUFA synthesis was not detected, consistent with the absence of ELOVL2 expression. An EFA ratio of 8:1 reduced 18:3n-3 conversion and enhanced 20:2n-6 synthesis compared to a 5:1 ratio. Here, [d5]9- and [d5]-13-hydroxyoctadecadienoic (HODE) and [13C]9- and [13C]13-hydroxyoctadecatrienoic acids (HOTrE) were the major labelled oxylipins in culture supernatants; labelled oxylipins ≥20 carbons were not detected. An EFA ratio of 8:1 suppressed 9- and 13-HOTrE synthesis, but there was no significant effect on 9- and 13-HODE synthesis. These findings suggest that partitioning of newly assimilated EFA between LCPUFA synthesis and hydroxyoctadecaenoic acid may be a metabolic branch point in T-cell EFA metabolism that has implications for understanding the effects of dietary fats on T lymphocyte function.

Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

The club cell, a small airway epithelial (SAE) cell, plays a central role in human lung host defense. We hypothesized that subpopulations of club cells with distinct functions may exist. The SAE of healthy nonsmokers and healthy cigarette smokers were evaluated by single-cell RNA sequencing, and unsupervised clustering revealed subpopulations of SCGCB1A1+KRT5loMUC5AC− club cells. Club cell heterogeneity was supported by evaluations of SAE tissue sections, brushed SAE cells, and in vitro air–liquid interface cultures. Three subpopulations included: (1) progenitor; (2) proliferating; and (3) effector club cells. The progenitor club cell population expressed high levels of mitochondrial, ribosomal proteins, and KRT5 relative to other club cell populations and included a differentiation branch point leading to mucous cell production. The small proliferating population expressed high levels of cyclins and proliferation markers. The effector club cell cluster expressed genes related to host defense, xenobiotic metabolism, and barrier functions associated with club cell function. Comparison of smokers vs. nonsmokers demonstrated that smoking limited the extent of differentiation of all three subclusters and altered SAM pointed domain-containing Ets transcription factor (SPDEF)-regulated transcription in the effector cell population leading to a change in the location of the branch point for mucous cell production, a potential explanation for the concomitant reduction in effector club cells and increase in mucous cells in smokers. These observations provide insights into both the makeup of human SAE club cell subpopulations and the smoking-induced changes in club cell biology.

U2 snRNA structure is influenced by SF3A and SF3B proteins but not by SF3B inhibitors

U2 snRNP is an essential component of the spliceosome. It is responsible for branch point recognition in the spliceosome A-complex via base-pairing of U2 snRNA with an intron to form the branch helix. Small molecule inhibitors target the SF3B component of the U2 snRNP and interfere with A-complex formation during spliceosome assembly. We previously found that the first SF3B inhibited-complex is less stable than A-complex and hypothesized that SF3B inhibitors interfere with U2 snRNA secondary structure changes required to form the branch helix. Using RNA chemical modifiers, we probed U2 snRNA structure in A-complex and SF3B inhibited splicing complexes. The reactivity pattern for U2 snRNA in the SF3B inhibited-complex is indistinguishable from that of A-complex, suggesting that they have the same secondary structure conformation, including the branch helix. This observation suggests SF3B inhibited-complex instability does not stem from an alternate RNA conformation and instead points to the inhibitors interfering with protein component interactions that normally stabilize U2 snRNP's association with an intron. In addition, we probed U2 snRNA in the free U2 snRNP in the presence of SF3B inhibitor and again saw no differences. However, increased protection of nucleotides upstream of Stem I in the absence of SF3A and SF3B proteins suggests a change of secondary structure at the very 5′ end of U2 snRNA. Chemical probing of synthetic U2 snRNA in the absence of proteins results in similar protections and predicts a previously uncharacterized extension of Stem I. Because this stem must be disrupted for SF3A and SF3B proteins to stably join the snRNP, the structure has the potential to influence snRNP assembly and recycling after spliceosome disassembly.

Convergence of Discrete Period Matrices and Discrete Holomorphic Integrals for Ramified Coverings of the Riemann Sphere

We consider the class of compact Riemann surfaces which are ramified coverings of the Riemann sphere $\hat {\mathbb {C}}$ ℂ ̂ . Based on a triangulation of this covering we define discrete (multivalued) harmonic and holomorphic functions. We prove that the corresponding discrete period matrices converge to their continuous counterparts. In order to achieve an error estimate, which is linear in the maximal edge length of the triangles, we suitably adapt the triangulations in a neighborhood of every branch point. Finally, we also prove a convergence result for discrete holomorphic integrals for our adapted triangulations of the ramified covering.

Branch point twist field form factors in the sine-Gordon model I: Breather fusion and entanglement dynamics

The quantum sine-Gordon model is the simplest massive interacting integrable quantum field theory whose two-particle scattering matrix is generally non-diagonal. As such, it is a model that has been extensively studied, especially in the context of the bootstrap program. In this paper we compute low particle-number form factors of a special local field known as the branch point twist field, whose correlation functions are building blocks for measures of entanglement. We consider the attractive regime where the theory possesses a particle spectrum consisting of a soliton, an antisoliton (of opposite U(1) charges) and several (neutral) breathers. In the breather sector we exploit the fusion procedure to compute form factors of heavier breathers from those of lighter ones. We apply our results to the study of the entanglement dynamics after a small mass quench and for short times. We show that in the presence of two or more breathers the von Neumann and Rényi entropies display undamped oscillations in time, whose frequencies are proportional to the even breather masses and whose amplitudes are proportional to the breather's one-particle form factor.

U(1) symmetry resolved entanglement in free 1+1 dimensional field theories via form factor bootstrap

We generalise the form factor bootstrap approach to integrable field theories with U(1) symmetry to derive matrix elements of composite branch-point twist fields associated with symmetry resolved entanglement entropies. The bootstrap equations are solved for the free massive Dirac and complex boson theories, which are the simplest theories with U(1) symmetry. We present the exact and complete solution for the bootstrap, including vacuum expectation values and form factors involving any type and arbitrarily number of particles. The non-trivial solutions are carefully cross-checked by performing various limits and by the application of the ∆-theorem. An alternative and compact determination of the novel form factors is also presented. Based on the form factors of the U(1) composite branch-point twist fields, we re-derive earlier results showing entanglement equipartition for an interval in the ground state of the two models.

Cellular Mn/Zn ratio influences phosphoglucomutase activity and capsule production in Streptococcus pneumoniae D39

Capsular polysaccharide (CPS) is a major virulence determinant for human pathogenic bacteria. Although the essential functional roles for CPS in bacterial virulence have been established, knowledge of how CPS production is regulated remains limited. Streptococcus pneumoniae (pneumococcus) CPS expression levels and overall thickness change in response to available oxygen and carbohydrate. These nutrients in addition to transition metal ions can vary significantly between host environmental niches and infection stage. Since pneumococcus must modulate CPS expression among various host niches during disease progression, we examined the impact of nutritional transition metal availability of manganese (Mn) and zinc (Zn) on CPS production. We demonstrate that increased Mn/Zn ratio increase CPS production via Mn-dependent activation of the phosphoglucomutase Pgm, an enzyme that functions at the branch point between glycolysis and the CPS biosynthetic pathway in a transcription-independent manner. Further, we find that the downstream CPS protein CpsB, a Mn-dependent phosphatase, does not promote aberrant dephosporylation of its target capsule-tyrosine kinase CpsD during Mn-stress. Together, these data reveal a direct role for cellular Mn/Zn ratios in the regulation of CPS biosynthesis via direct activation of Pgm. We propose a multilayer mechanism used by the pneumococcus in regulating CPS levels across various host niches. IMPORTANCE Evolving evidence strongly indicates that maintenance of metal homeostasis is essential for establishing colonization and continued growth of bacterial pathogens in the vertebrate host. In this study, we demonstrate the impact of cellular manganese/zinc (Mn/Zn) ratios on bacterial capsular polysaccharide (CPS) production, an important virulence determinant of many human pathogenic bacteria, including Streptococcus pneumoniae. We show that higher Mn/Zn ratios increase CPS production via Mn-dependent activation of the phosphoglucomutase Pgm, an enzyme that functions at the branch point between glycolysis and the CPS biosynthetic pathway. The findings provide a direct role for Mn/Zn homeostasis in the regulation of CPS expression levels and further support the ability for metal cations to act as important cellular signaling mediators in bacteria.