The DEAD-box ATPase Dbp10/DDX54 initiates peptidyl transferase center formation during 60S ribosome biogenesis.

DEAD-box ATPases play crucial roles in guiding rRNA restructuring events during the biogenesis of large (60S) ribosomal subunits, but their precise molecular functions are currently unknown. In this study, we present cryo-EM reconstructions of nucleolar pre-60S intermediates that reveal an unexpected, alternate secondary structure within the nascent peptidyl-transferase-center (PTC). Our analysis of three sequential nucleolar pre-60S intermediates reveals that the DEAD-box ATPase Dbp10/DDX54 remodels this alternate base pairing and enables the formation of the rRNA junction that anchors the mature form of the universally conserved PTC A-loop. Post-catalysis, Dbp10 captures rRNA helix H61, initiating the concerted exchange of biogenesis factors during late nucleolar 60S maturation. Our findings show that Dbp10 activity is essential for the formation of the ribosome active site and reveal how this function is integrated with subsequent assembly steps to drive the biogenesis of the large ribosomal subunit.

The DEAD-box ATPase Dbp10/DDX54 initiates peptidyl transferase center formation during 60S ribosome biogenesis.

DEAD-box ATPases play crucial roles in guiding rRNA restructuring events during the biogenesis of large (60S) ribosomal subunits, but their precise molecular functions are currently unknown. In this study, we present cryo-EM reconstructions of nucleolar pre-60S intermediates that reveal an unexpected, alternate secondary structure within the nascent peptidyl-transferase-center (PTC). Our analysis of three sequential nucleolar pre-60S intermediates reveals that the DEAD-box ATPase Dbp10/DDX54 remodels this alternate base pairing and enables the formation of the rRNA junction that anchors the mature form of the universally conserved PTC A-loop. Post-catalysis, Dbp10 captures rRNA helix H61, initiating the concerted exchange of biogenesis factors during late nucleolar 60S maturation. Our findings show that Dbp10 activity is essential for the formation of the ribosome active site and reveal how this function is integrated with subsequent assembly steps to drive the biogenesis of the large ribosomal subunit.

A comprehensive landscape of 60S ribosome biogenesis factors.

Eukaryotic ribosome biogenesis is facilitated and regulated by numerous ribosome biogenesis factors (RBFs). High-resolution cryoelectron microscopy (cryo-EM) maps have defined the molecular interactions of RBFs during maturation, but many transient and dynamic interactions, particularly during early assembly, remain uncharacterized. Using quantitative proteomics and crosslinking coupled to mass spectrometry (XL-MS) data from an extensive set of pre-ribosomal particles, we derive a comprehensive and time-resolved interaction map of RBF engagement during 60S maturation. We localize 22 previously unmapped RBFs to specific biogenesis intermediates and validate our results by mapping the catalytic activity of the methyltransferases Bmt2 and Rcm1 to their predicted nucleolar 60S intermediates. Our analysis reveals the interaction sites for the RBFs Noc2 and Ecm1 and elucidates the interaction map and timing of 60S engagement by the DEAD-box ATPases Dbp9 and Dbp10. Our data provide a powerful resource for future studies of 60S ribosome biogenesis.

Structures of FHOD1-Nesprin1/2 complexes reveal alternate binding modes for the FH3 domain of formins.

The nuclear position in eukaryotes is controlled by a nucleo-cytoskeletal network, critical in cell differentiation, division, and movement. Forces are transmitted through conserved Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes that traverse the nuclear envelope and engage on either side of the membrane with diverse binding partners. Nesprin-2-giant (Nes2G), a LINC element in the outer nuclear membrane, connects to the actin directly as well as through FHOD1, a formin primarily involved in actin bundling. Here, we report the crystal structure of Nes2G bound to FHOD1 and show that the presumed G-binding domain of FHOD1 is rather a spectrin repeat (SR) binding enhancer for the neighboring FH3 domain. The structure reveals that SR binding by FHOD1 is likely not regulated by the diaphanous-autoregulatory domain helix of FHOD1. Finally, we establish that Nes1G also has one FHOD1 binding SR, indicating that these abundant, giant Nesprins have overlapping functions in actin-bundle recruitment for nuclear movement.

Structural Analysis of Different LINC Complexes Reveals Distinct Binding Modes.

Linker of nucleoskeleton and cytoskeleton (LINC) complexes are molecular tethers that span the nuclear envelope (NE) and physically connect the nucleus to the cytoskeleton. They transmit mechanical force across the NE in processes such as nuclear anchorage, nuclear migration, and homologous chromosome pairing during meiosis. LINC complexes are composed of KASH proteins traversing the outer nuclear membrane, and SUN proteins crossing the inner nuclear membrane. Humans have several SUN- and KASH-containing proteins, yet what governs their proper engagement is poorly understood. To investigate this question, we solved high resolution crystal structures of human SUN2 in complex with the KASH-peptides of Nesprin3, Nesprin4, and KASH5. In comparison to the published structures of SUN2-KASH1/2 we observe alternative binding modes for these KASH peptides. While the core interactions between SUN and the C-terminal residues of the KASH peptide are similar in all five complexes, the extended KASH-peptide adopts at least two different conformations. The much-improved resolution allows for a more detailed analysis of other elements critical for KASH interaction, including the KASH-lid and the cation loop, and a possible self-locked state for unbound SUN. In summary, we observe distinct differences between the examined SUN-KASH complexes. These differences may have an important role in regulating the SUN-KASH network.

Recombinant Purification of the Periplasmic Portion of the LINC Complex.

Recombinant expression of proteins and their complexes is the routine laboratory procedure to generate pure reagents for biochemical and structural studies. Here we present the standard procedure developed in our lab for the production of milligram quantities of stoichiometric SUN-KASH complexes. The protocol was specifically developed for the purification of the periplasmic portion of LINC complexes.

Localización inusual del hidradenoma papilífero / Unusual location of the papilliform hydradenoma

El hidradenoma papilífero es una tumoración benigna, rara, generalmente localizada en la región vulvar o perineal siendo la región perianal una localización poco frecuente. Su asociación con enfermedades de transmisión sexual y el carcinoma ductal es controvertida; es importante realizar el estudio histopatológico de cualquier tumoración en esta zona. Se reporta el caso de una paciente de 49 años de edad, con un nódulo en la región perianal de 2 años de evolución; el estudio histopatológico confirmó el diagnóstico de hidradenoma papilífero. (AU)

The papilliferous hidradenoma is a benign, rare tumor, usually located in the vulvar or perineal region and the perianal region is a rare site. Its association with sexually transmitted diseases and ductal carcinoma is controversial. It is important to perform the histopathological study of any tumor in this area. The case of a 49-year-old patient with a nodule in the perianal region of 2 years of evolution is reported. The histopathological study confirmed the diagnosis of papilliferous hidradenoma.

LINC complexes promote homologous recombination in part through inhibition of nonhomologous end joining.

The Caenorhabditis elegans SUN domain protein, UNC-84, functions in nuclear migration and anchorage in the soma. We discovered a novel role for UNC-84 in DNA damage repair and meiotic recombination. Loss of UNC-84 leads to defects in the loading and disassembly of the recombinase RAD-51. Similar to mutations in Fanconi anemia (FA) genes, unc-84 mutants and human cells depleted of Sun-1 are sensitive to DNA cross-linking agents, and sensitivity is rescued by the inactivation of nonhomologous end joining (NHEJ). UNC-84 also recruits FA nuclease FAN-1 to the nucleoplasm, suggesting that UNC-84 both alters the extent of repair by NHEJ and promotes the processing of cross-links by FAN-1. UNC-84 interacts with the KASH protein ZYG-12 for DNA damage repair. Furthermore, the microtubule network and interaction with the nucleoskeleton are important for repair, suggesting that a functional linker of nucleoskeleton and cytoskeleton (LINC) complex is required. We propose that LINC complexes serve a conserved role in DNA repair through both the inhibition of NHEJ and the promotion of homologous recombination at sites of chromosomal breaks.

The Caenorhabditis elegans Protein FIC-1 Is an AMPylase That Covalently Modifies Heat-Shock 70 Family Proteins, Translation Elongation Factors and Histones.

Protein AMPylation by Fic domain-containing proteins (Fic proteins) is an ancient and conserved post-translational modification of mostly unexplored significance. Here we characterize the Caenorhabditis elegans Fic protein FIC-1 in vitro and in vivo. FIC-1 is an AMPylase that localizes to the nuclear surface and modifies core histones H2 and H3 as well as heat shock protein 70 family members and translation elongation factors. The three-dimensional structure of FIC-1 is similar to that of its human ortholog, HYPE, with 38% sequence identity. We identify a link between FIC-1-mediated AMPylation and susceptibility to the pathogen Pseudomonas aeruginosa, establishing a connection between AMPylation and innate immunity in C. elegans.

Purification and Structural Analysis of SUN and KASH Domain Proteins.

Molecular tethers span the nuclear envelope to mechanically connect the cytoskeleton and nucleoskeleton. These bridge-like tethers, termed linkers of nucleoskeleton and cytoskeleton (LINC) complexes, consist of SUN proteins at the inner nuclear membrane and KASH proteins at the outer nuclear membrane. LINC complexes are central to a variety of cell activities including nuclear positioning and mechanotransduction, and LINC-related abnormalities are associated with a spectrum of tissue-specific diseases, termed laminopathies or envelopathies. Protocols used to study the biochemical and structural characteristics of core elements of SUN-KASH complexes are described here to facilitate further studies in this new field of cell biology.

Differential gender-related susceptibility to learning and memory deficits in mice submitted to neonatal freezing microgyria model.

Sexual dimorphism during mammalian neural development seems to contribute to differential gender-related incidence in malformations of cortical development in both humans and rodents. Here we investigated the existence of differential gender-related susceptibility to learning and memory deficits and brain injury severity in mice submitted to a microgyria model. Newborn male and female C57BL/6 mice (P0) were submitted to a unilateral freezing lesion (FL) using a cooled steel probe, placed over the right midline anteroposterior plane. Mice were allowed to survive for 12-14 weeks and then were submitted to behavioral tasks and brain morphological analyses. Injured mice from both genders did not present gross locomotor alterations, and the freezing lesion resulted in similar brain damage in male and female mice. Additionally, a selective disruption in the short-term social recognition memory was observed in injured male mice while the long-term inhibitory avoidance memory was not affected by both the factors. These results indicate a reduced susceptibility of female to short-term social-memory deficits induced by neonatal model of microgyria in mice, suggesting that the cognitive deficits induced by freezing lesions in rodents may not be entirely related to the severity of brain injury.