Centromere identity is dependent on nuclear spatial organization

The establishment of centromere-specific CENP-A chromatin is influenced by epigenetic and genetic processes. Central domain sequences from fission yeast centromeres are preferred substrates for CENP-ACnp1 incorporation, but their use is context dependent, requiring adjacent heterochromatin. CENP-ACnp1 overexpression bypasses heterochromatin dependency, suggesting heterochromatin ensures exposure to conditions or locations permissive for CENP-ACnp1 assembly. Centromeres cluster around spindle-pole bodies (SPBs). We show that heterochromatin-bearing minichromosomes localize close to SPBs, consistent with this location promoting CENP-ACnp1 incorporation. We demonstrate that heterochromatin-independent de novo CENP-ACnp1 chromatin assembly occurs when central domain DNA is placed near, but not far from, endogenous centromeres or neocentromeres. Moreover, direct tethering of central domain DNA at SPBs permits CENP-ACnp1 assembly, suggesting that the nuclear compartment surrounding SPBs is permissive for CENP-ACnp1 incorporation because target sequences are exposed to high levels of CENP-ACnp1 and associated assembly factors. Thus, nuclear spatial organization is a key epigenetic factor that influences centromere identity.

P.123 Severe DNM1 Encephalopathy with Dysmyelination due to Recurrent Splice Site Pathogenic Variant

Background: Patients with DNM1-encephalopathy almost exclusively have missense variants, mostly in the GTPase domain of DNM1. Delayed myelination has been reported in at least three patients with DNM1-encephalopathy, all with missense mutations in the DNM1 central domain. Only one DNM1 splice-site variant has previously been reported, and the authors questioned whether the variant accounted for all aspects of the patient’s phenotype. Methods: Case-Report. Results: Our patient had hypotonia and brief multifocal tonic seizures from age-1-month. He still has profound global developmental delay, daily seizures and microcephaly. MRI-Brain at age-21-months showed T2 hyperintensity in the bilateral periventricular and subcortical white matter; spectroscopy showed a questionable lactate peak and an elevated choline peak relative to N-acetylaspartate. Clinical gene-panel identified a heterozygous de novo pathogenic variant in intron 9 of DNM1 (c.1197-8G > A; IVS9- 8G>A). In-silico tools categorized this variant as deleterious secondary to a splicing defect. RT-PCR analysis on peripheral blood was unsuccessful as DNM1 expression is extremely low outside of the brain. Conclusions: Our patient carried the same DNM1 variant previously reported, indicating this is a recurrent pathogenic splice-site variant. The spectroscopic abnormalities suggest a possible element of demyelination in DNM1 variants of the central domain, though the mechanism remains unclear.

Critically analysis of Shobasakthi Box Story Book

Shobashakti is one of the most important migrant creators of today's Eelam. He has created novels including Gorilla, M and BOX. This paper deals with BOX. The new day brings to the fore the geological and social and historical references to the village of Palankulam, the central domain of the story, and the various war-related situations of Eelam. This study reveals them critically. Moreover, the story is told under a separate title from Mayyakakatha to Forty in the novel. In addition, the final version of the story has been set up. Under some headings subsection symmesis sub-copies 1 and 2 are also included. The subsection of the subcopies is centered on the statement of the characters and the textual record of the heart queen and the text language record of Roman Bhaktadas are the new techniques and methods in the new system. They are also discussed in the article.

Kinetoplastid kinetochore proteins KKT2 and KKT3 have unique centromere localization domains

The kinetochore is the macromolecular protein complex that assembles onto centromeric DNA and binds spindle microtubules. Evolutionarily divergent kinetoplastids have an unconventional set of kinetochore proteins. It remains unknown how kinetochores assemble at centromeres in these organisms. Here, we characterize KKT2 and KKT3 in the kinetoplastid parasite Trypanosoma brucei. In addition to the N-terminal kinase domain and C-terminal divergent polo boxes, these proteins have a central domain of unknown function. We show that KKT2 and KKT3 are important for the localization of several kinetochore proteins and that their central domains are sufficient for centromere localization. Crystal structures of the KKT2 central domain from two divergent kinetoplastids reveal a unique zinc-binding domain (termed the CL domain for centromere localization), which promotes its kinetochore localization in T. brucei. Mutations in the equivalent domain in KKT3 abolish its kinetochore localization and function. Our work shows that the unique central domains play a critical role in mediating the centromere localization of KKT2 and KKT3.

Shape changes and cooperativity in the folding of the central domain of the 16S ribosomal RNA

Both the small and large subunits of the ribosome, the molecular machine that synthesizes proteins, are complexes of ribosomal RNAs (rRNAs) and a number of proteins. In bacteria, the small subunit has a single 16S rRNA whose folding is the first step in its assembly. The central domain of the 16S rRNA folds independently, driven either by Mg2+ ions or by interaction with ribosomal proteins. To provide a quantitative description of ion-induced folding of the ∼350-nucleotide rRNA, we carried out extensive coarse-grained molecular simulations spanning Mg2+ concentration between 0 and 30 mM. The Mg2+ dependence of the radius of gyration shows that globally the rRNA folds cooperatively. Surprisingly, various structural elements order at different Mg2+ concentrations, indicative of the heterogeneous assembly even within a single domain of the rRNA. Binding of Mg2+ ions is highly specific, with successive ion condensation resulting in nucleation of tertiary structures. We also predict the Mg2+-dependent protection factors, measurable in hydroxyl radical footprinting experiments, which corroborate the specificity of Mg2+-induced folding. The simulations, which agree quantitatively with several experiments on the folding of a three-way junction, show that its folding is preceded by formation of other tertiary contacts in the central junction. Our work provides a starting point in simulating the early events in the assembly of the small subunit of the ribosome.

Gulf of Aden spreading does not conform to triple-junction formation

The Gulf of Aden represents an evolving example of a juvenile ocean system and is considered the most evolved rift arm of the Afar triple junction. We have undertaken analysis of recent coupled satellite and marine potential-field data to understand the first-order crustal architecture along the entire length of the gulf. Our interpretation suggests the Gulf of Aden has three domains with distinct free-air gravity and magnetic characteristics. These domains record a progression from active seafloor spreading in the eastern domain, through isolated and discontinuous spreading segments in the central domain, to active continental rifting in the western domain immediately adjacent to the Afar triple junction. Forward models suggest the presence of transitional crust, which displays linear magnetic stripe–like anomalies that bound oceanic stripes in the central domain and covering the majority of the western domain. Magnetic anomalies differ from magnetic stripes sensu stricto because they are discontinuous and cannot be correlated along the length of the gulf. Detection of northwest-southeast extension in the central domain based on magnetic stripe orientation is inconsistent with the regional northeast-southwest extension. Our observations reflect heterogeneous opening of the Gulf of Aden basins, in which spreading is migrating toward Afar as a series of isolated spreading segments, rather than initiating at the junction as proposed by classical platetectonic theory. This mechanism of ocean initiation is inconsistent with transtensional models that involve wholesale tearing of continental crust and contradicts conceptual models that rely on the Afar plume in initiating or driving the extension.

Znc2 module of RAG1 contributes towards structure-specific nuclease activity of RAGs

Recombination activating genes (RAGs), consisting of RAG1 and RAG2 have ability to perform spatially and temporally regulated DNA recombination in a sequence specific manner. Besides, RAGs also cleave at non-B DNA structures and are thought to contribute towards genomic rearrangements and cancer. The nonamer binding domain of RAG1 binds to the nonamer sequence of the signal sequence during V(D)J recombination. However, deletion of NBD did not affect RAG cleavage on non-B DNA structures. In the present study, we investigated the involvement of other RAG domains when RAGs act as a structure-specific nuclease. Studies using purified central domain (CD) and C-terminal domain (CTD) of the RAG1 showed that CD of RAG1 exhibited high affinity and specific binding to heteroduplex DNA, which was irrespective of the sequence of single-stranded DNA, unlike CTD which showed minimal binding. Furthermore, we show that ZnC2 of RAG1 is crucial for its binding to DNA structures as deletion and point mutations abrogated the binding of CD to heteroduplex DNA. Our results also provide evidence that unlike RAG cleavage on RSS, central domain of RAG1 is sufficient to cleave heteroduplex DNA harbouring pyrimidines, but not purines. Finally, we show that a point mutation in the DDE catalytic motif is sufficient to block the cleavage of CD on heteroduplex DNA. Therefore, in the present study we demonstrate that the while ZnC2 module in central domain of RAG1 is required for binding to non-B DNA structures, active site amino acids are important for RAGs to function as a structure-specific nuclease.

Cryo-EM structure of yeast 90S preribosome at 3.4 Å resolution

Previously, we determined a cryo-EM structure of Saccharomyces cerevisiae 90S preribosome obtained after depletion of the RNA helicase Mtr4 at 4.5 Å resolution (Sun et al., 2017). The 90S preribosome is an early assembly intermediate of small ribosomal subunit. Here, the structure was improved to 3.4 Å resolution and reveals many previously unresolved structures and interactions, in particular around the central domain of 18S rRNA. The central domain adopts a closed conformation in our structure, in contrast to an open conformation in another high-resolution structure of S. cerevisiae 90S. The new model of 90S would serve as a better reference for investigation of the assembly mechanism of small ribosomal subunit.

The central domain of cardiac ryanodine receptor governs channel activation, regulation, and stability

Structural analyses identified the central domain of ryanodine receptor (RyR) as a transducer converting conformational changes in the cytoplasmic platform to the RyR gate. The central domain is also a regulatory hub encompassing the Ca2+-, ATP-, and caffeine-binding sites. However, the role of the central domain in RyR activation and regulation has yet to be defined. Here, we mutated five residues that form the Ca2+ activation site and 10 residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site. We also generated eight disease-associated mutations within the central domain of RyR2. We determined the effect of these mutations on Ca2+, ATP, and caffeine activation and Mg2+ inhibition of RyR2. Mutating the Ca2+ activation site markedly reduced the sensitivity of RyR2 to Ca2+ and caffeine activation. Unexpectedly, Ca2+ activation site mutation E3848A substantially enhanced the Ca2+-independent basal activity of RyR2, suggesting that E3848A may also affect the stability of the closed state of RyR2. Mutations in the Ca2+ activation site also abolished the effect of ATP/caffeine on the Ca2+-independent basal activity, suggesting that the Ca2+ activation site is also a critical determinant of ATP/caffeine action. Mutating residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site significantly altered Ca2+ and caffeine activation and reduced Mg2+ inhibition. Furthermore, disease-associated RyR2 mutations within the central domain significantly enhanced Ca2+ and caffeine activation and reduced Mg2+ inhibition. Our data demonstrate that the central domain plays an important role in channel activation, channel regulation, and closed state stability.