Synthesis of 5′-Thiamine-Capped RNA

RNA 5′-modifications are known to extend the functional spectrum of ribonucleotides. In recent years, numerous non-canonical 5′-modifications, including adenosine-containing cofactors from the group of B vitamins, have been confirmed in all kingdoms of life. The structural component of thiamine adenosine triphosphate (thiamine-ATP), a vitamin B1 derivative found to accumulate in Escherichia coli and other organisms in response to metabolic stress conditions, suggests an analogous function as a 5′-modification of RNA. Here, we report the synthesis of thiamine adenosine dinucleotides and the preparation of pure 5′-thiamine-capped RNAs based on phosphorimidazolide chemistry. Furthermore, we present the incorporation of thiamine-ATP and thiamine adenosine diphosphate (thiamine-ADP) as 5′-caps of RNA by T7 RNA polymerase. Transcripts containing the thiamine modification were modified specifically with biotin via a combination of thiazole ring opening, nucleophilic substitution and copper-catalyzed azide-alkyne cycloaddition. The highlighted methods provide easy access to 5′-thiamine RNA, which may be applied in the development of thiamine-specific RNA capture protocols as well as the discovery and confirmation of 5′-thiamine-capped RNAs in various organisms.

Regulation of an iron-dependent repressor by tryptophan availability attenuates transcription of the tryptophan salvage genes in Chlamydia trachomatis

The trp operon of Chlamydia trachomatis has a genetic organization that is distinct from other model bacteria. The operon contains the trpR open-reading frame (ORF), an intergenic region (IGR), and the trpB and trpA ORFs. TrpR mediates tryptophan-dependent regulation of the operon from the major promoter upstream of trpR(PtrpR). We recently reported that trpBA is additionally regulated by the iron-dependent repressor YtgR via an operator sequence within the IGR upstream of an alternative promoter for TrpR-independent trpBA expression (PtrpBA). Here we report that YtgR repression of PtrpBA is also dependent on tryptophan levels via a rare triple-tryptophan motif (WWW) in the N-terminal permease domain of the YtgCR precursor. Tryptophan limitation inhibits translation at the WWW motif and subsequently promotes transcription termination of ytgCR in a Rho-independent manner. This regulatory schematic resembles mechanisms of transcriptional attenuation for trp operons described in model bacteria, such as cis-attenuation by TrpL in E. coli or trans-attenuation by TRAP in B. subtilis. YtgR performs an analogous function by sensing both iron and tryptophan levels, with the latter highlighting the unique strategy of Chlamydia to retain a trans-attenuator mechanism for regulating expression of the trpRBA operon.

Degeneracy in hippocampal physiology and plasticity

ABSTRACTDegeneracy, defined as the ability of structurally disparate elements to perform analogous function, has largely been assessed from the perspective of maintaining robustness of physiology or plasticity. How does the framework of degeneracy assimilate into an encoding system where the ability to change is an essential ingredient for storing new incoming information? Could degeneracy maintain the balance between the apparently contradictory goals of the need to change for encoding and the need to resist change towards maintaining homeostasis? In this review, we explore these fundamental questions with the mammalian hippocampus as an example encoding system. We systematically catalog lines of evidence, spanning multiple scales of analysis, that demonstrate the expression of degeneracy in hippocampal physiology and plasticity. We assess the potential of degeneracy as a framework to achieve the conjoint goals of encoding and homeostasis without cross-interferences. We postulate that biological complexity, involving interactions among the numerous parameters spanning different scales of analysis, could establish disparate routes towards accomplishing these conjoint goals. These disparate routes then provide several degrees of freedom to the encoding-homeostasis system in accomplishing its tasks in an input- and state-dependent manner. Finally, the expression of degeneracy spanning multiple scales offers an ideal reconciliation to several outstanding controversies, through the recognition that the seemingly contradictory disparate observations are merely alternate routes that the system might recruit towards accomplishment of its goals. Against the backdrop of the ubiquitous prevalence of degeneracy and its strong links to evolution, it is perhaps apt to add a corollary to Theodosius Dobzhansky’s famous quote and state “nothing in physiology makes sense except in the light of degeneracy”.HighlightsDegeneracy is the ability of structurally distinct elements to yield similar functionWe postulate a critical role for degeneracy in the emergence of stable encoding systemsWe catalog lines of evidence for the expression of degeneracy in the hippocampusWe suggest avenues for research to explore degeneracy in stable encoding systemsDobzhansky wrote: “nothing in biology makes sense except in the light of evolution”A corollary: “nothing in physiology makes sense except in the light of degeneracy”

Myogenic Differentiation from MYOGENIN-Mutated Human iPS Cells by CRISPR/Cas9

It is well known that myogenic regulatory factors encoded by the Myod1 family of genes have pivotal roles in myogenesis, with partially overlapping functions, as demonstrated for the mouse embryo. Myogenin-mutant mice, however, exhibit severe myogenic defects without compensation by other myogenic factors. MYOGENIN might be expected to have an analogous function in human myogenic cells. To verify this hypothesis, we generated MYOGENIN-mutated human iPS cells by using CRISPR/Cas9 genome-editing technology. Our results suggest that MYOD1-independent or MYOD1-dependent mechanisms can compensate for the loss of MYOGENIN and that these mechanisms are likely to be crucial for regulating skeletal muscle differentiation and formation.

Auxilin facilitates membrane traffic in the early secretory pathway

Coat protein complexes contain an inner shell that sorts cargo and an outer shell that helps deform the membrane to give the vesicle its shape. There are three major types of coated vesicles in the cell: COPII, COPI, and clathrin. The COPII coat complex facilitates vesicle budding from the endoplasmic reticulum (ER), while the COPI coat complex performs an analogous function in the Golgi. Clathrin-coated vesicles mediate traffic from the cell surface and between the trans-Golgi and endosome. While the assembly and structure of these coat complexes has been extensively studied, the disassembly of COPII and COPI coats from membranes is less well understood. We describe a proteomic and genetic approach that connects the J-domain chaperone auxilin, which uncoats clathrin-coated vesicles, to COPII and COPI coat complexes. Consistent with a functional role for auxilin in the early secretory pathway, auxilin binds to COPII and COPI coat subunits. Furthermore, ER–Golgi and intra-Golgi traffic is delayed at 15°C in swa2Δ mutant cells, which lack auxilin. In the case of COPII vesicles, we link this delay to a defect in vesicle fusion. We propose that auxilin acts as a chaperone and/or uncoating factor for transport vesicles that act in the early secretory pathway.

The Chowla–Selberg formula for abelian CM fields and Faltings heights

In this paper we establish a Chowla–Selberg formula for abelian CM fields. This is an identity which relates values of a Hilbert modular function at CM points to values of Euler’s gamma function ${\rm\Gamma}$ and an analogous function ${\rm\Gamma}_{2}$ at rational numbers. We combine this identity with work of Colmez to relate the CM values of the Hilbert modular function to Faltings heights of CM abelian varieties. We also give explicit formulas for products of exponentials of Faltings heights, allowing us to study some of their arithmetic properties using the Lang–Rohrlich conjecture.

Structural analysis of the C-terminal domain of the spliceosomal helicase Prp22

Splicing of pre-mRNA requires the activity of at least eight different DEAD/H-box proteins that are involved in distinct steps of the splicing process. These proteins are driving the spliceosomal machinery by ATP-dependent unwinding of dsRNA and/or disrupting protein-RNA complexes. The spliceosomal DEAH-box proteins Prp2, Prp16, Prp22 and Prp43 share homologous C-terminal domains (CTD). We have determined the crystal structure of the CTD of human Prp22 by means of MAD. The fold of the human Prp22-CTD closely resembles that of the yeast Prp43-CTD. The similarity of these helicase-associated CTDs to the winged-helix and ratchet domains of the DNA helicase Hel308 suggests an analogous function in dsRNA binding and unwinding. Here, we also demonstrate that the CTD has a significant impact on the ATPase activity of yPrp22 in vitro. Homology modeling of the CTDs of hPrp2, hPrp16 and hPrp43 suggests that the CTDs of spliceosomal helicases contain conserved positively charged patches on their surfaces representing a common RNA-binding surface as well as divergent regions most likely mediating specific interactions with different proteins of the spliceosome.

The dual J* of the James space has cotype 2 and the Gordon-Lewis property

We prove the result in the title. More generally we consider the Banach space υp, of all sequences (xn) of scalars such thatwhere the supremum runs over all increasing sequences n1 ≤ n2 ≤ …. We show that is of cotype 2 if p ≽ 2 and of cotype p′, where 1/p′ + 1/p = 1, if p ≤ 2. Similar results are obtained for the analogous function spaces.