Three-dimensional organisation of RNA-processing machinery in avian growing oocyte nucleus.

Chromosome architecture and assorted nuclear compartments play an essential role in RNA transcription and processing. Growing vertebrate oocytes represent an advantageous model to study the principles of nuclear structure and function. In this article, the data on three-dimensional (3D) organisation of intact and non-deformed oocyte nuclei (germinal vesicles) in four species of birds (domestic chicken, Japanese quail, rock pigeon and chaffinch) obtained by confocal laser scanning microscopy are presented. The nucleus of the growing avian oocyte has an unusual structure of RNA processing machinery. Germinal vesicles from any of the avian species studied, with the exception of the rock pigeon, are characterized by absence of extrachromosomal RNA-enriched nuclear bodies including Cajal bodies, histone locus bodies and interchromatin granule clusters. The absence of Cajal bodies and histone locus bodies in chicken oocytes correlated with the inactivation of nucleolus organizer and clustered histone genes. Splicing factors such as SR-protein SC35 accumulated in chromosome-associated domains that were classified as complex loops (terminal giant and lumpy loops). Formation of such depot in avian oocyte nuclei is supposed to be nucleated by transcripts of non-coding tandem repeats. The results obtained strongly support a model of RNA-mediated nuclear domains formation.

Non-canonical Cajal bodies form in the nucleus of late stage avian oocytes lacking functional nucleolus.

In the somatic cell nucleus, there are several universal domains such as nucleolus, SC35-domains, Cajal bodies (CBs) and histone locus bodies (HLBs). Among them, CBs were described more than 100 years ago; however, we still do not have a final understanding of their nature and biological significance. The giant nucleus of avian and amphibian growing oocytes represents an advantageous model for analysis of functions and biogenesis of various nuclear domains. Nevertheless, in large-sized avian oocytes that contain transcriptionally active lampbrush chromosomes, CB-like organelles have not been identified yet. Here we demonstrate that in the pigeon (Columba livia) oocyte nucleus, characterized by absence of any functional nucleoli, extrachromosomal spherical bodies contain TMG-capped spliceosomal snRNAs, core proteins of Sm snRNPs and the protein coilin typical for CBs, but not splicing factor SC35 nor the histone pre-mRNA 3'-end processing factor symplekin. The results establish that coilin-rich nuclear organelles in pigeon late-stage oocyte are not the equivalents of HLBs but belong to a group of CBs. At the same time, they do not contain the snoRNP/scaRNP protein fibrillarin involved in 2'-O-methylation of snoRNAs and snRNAs. Thus, the nucleus of late-stage pigeon oocytes houses CB-like organelles that have an unusual molecular composition and are implicated in the snRNP biogenesis pathway. These data demonstrate that snRNP-rich non-canonical CBs can form in the absence of nucleolus. We argue that pigeon oocytes represent a new promising model to investigate CB modular organization, functions and formation mechanism.

Low-voltage scanning electron microscopy study of lampbrush chromosomes and nuclear bodies in avian and amphibian oocytes.

Nucleus is a highly compartmentalized part of the cell where the key processes of genome functionality are realized through the formation of non-membranous nuclear domains. Physically nuclear domains appear as liquid droplets with different viscosity stably maintained throughout the interphase or during the long diplotene stage of meiosis. Since nuclear body surface represents boundary between two liquid phases, the ultrastructural surface topography of nuclear domains is of an outstanding interest. The aim of this study was to examine ultrathin surface topography of the amphibian and avian oocyte nuclear structures such as lampbrush chromosomes, nucleoli, histone-locus bodies, Cajal body-like bodies, and the interchromatin granule clusters via low-voltage scanning electron microscopy. Our results demonstrate that nuclear bodies with similar molecular composition may differ dramatically in the surface topography and vice versa, nuclear bodies that do not share common molecular components may possess similar topographical characteristics. We also have analyzed surface distribution of particular nuclear antigens (double stranded DNA, coilin and splicing snRNA) using indirect immunogold labeling with subsequent secondary electron detection of gold nanoparticles. We suggest that ultrastructural surface morphology reflects functional status of a nuclear body.

Genetic Spectrum of Idiopathic Restrictive Cardiomyopathy Uncovered by Next-Generation Sequencing.

BACKGROUND: Cardiomyopathies represent a rare group of disorders often of genetic origin. While approximately 50% of genetic causes are known for other types of cardiomyopathies, the genetic spectrum of restrictive cardiomyopathy (RCM) is largely unknown. The aim of the present study was to identify the genetic background of idiopathic RCM and to compile the obtained genetic variants to the novel signalling pathways using in silico protein network analysis. PATIENTS AND METHODS: We used Illumina MiSeq setup to screen for 108 cardiomyopathy and arrhythmia-associated genes in 24 patients with idiopathic RCM. Pathogenicity of genetic variants was classified according to American College of Medical Genetics and Genomics classification. RESULTS: Pathogenic and likely-pathogenic variants were detected in 13 of 24 patients resulting in an overall genotype-positive rate of 54%. Half of the genotype-positive patients carried a combination of pathogenic, likely-pathogenic variants and variants of unknown significance. The most frequent combination included mutations in sarcomeric and cytoskeletal genes (38%). A bioinformatics approach underlined the mechanotransducing protein networks important for RCM pathogenesis. CONCLUSIONS: Multiple gene mutations were detected in half of the RCM cases, with a combination of sarcomeric and cytoskeletal gene mutations being the most common. Mutations of genes encoding sarcomeric, cytoskeletal, and Z-line-associated proteins appear to have a predominant role in the development of RCM.

Congenital heart defects are rarely caused by mutations in cardiac and smooth muscle actin genes.

BACKGROUND: Congenital heart defects (CHDs) often have genetic background due to missense mutations in cardiomyocyte-specific genes. For example, cardiac actin was shown to be involved in pathogenesis of cardiac septum defects and smooth muscle actin in pathogenesis of aortic aneurysm in combination with patent ductus arteriosus (PDA). In the present study, we further searched for mutations in human α-cardiac actin (ACTC1) and smooth muscle α-actin (ACTA2) genes as a possible cause of atrial septum defect type II (ASDII) and PDA. FINDINGS: Total genomic DNA was extracted from peripheral blood of 86 individuals with ASDs and 100 individuals with PDA. Coding exons and flanking intron regions of ACTC1 (NM_005159.4) and ACTA2 (NM_001613) were amplified by PCR with specific primers designed according to the corresponding gene reference sequences. PCR fragments were directly sequenced and analyzed. Sequence analysis of ACTC1 and ACTA2 did not identify any nucleotide changes that altered the coding sense of the genes. In ACTC1 gene, we were able to detect one previously described nucleotide polymorphism (rs2307493) resulting in a synonymous substitution. The frequency of this SNP was similar in the study and control group, thus excluding it from the possible disease-associated variants. CONCLUSIONS: Our results confirmed that the mutations in ACTC1 gene are rare (at least <1%) cause of ASDII. Mutations in ACTA2 gene were not detected in patients with PDA, thus being excluded from the list of frequent PDA-associated genetic defects.