Congenital heart disease (CHD) is a leading cause of death in children <1 year of age. Despite intense effort in the last 10 years, most CHDs (~70%) still have an unknown etiology. Conotruncal based defects, such as Tetralogy of Fallot (TOF), a common complex of devastating heart defects, typically requires surgical intervention in the first year of life. We reported that the noncoding transcriptome in myocardial tissue from children with TOF is characterized by significant variation in levels of expression of noncoding RNAs, and more specifically, a significant reduction in 12 small cajal body-associated RNAs (scaRNAs) in the right ventricle. scaRNAs are essential for the biochemical modification and maturation of small nuclear RNAs (spliceosomal RNAs), which in turn are critical components of the spliceosome. This is particularly important because we also documented that splicing of mRNAs that are critical for heart development was dysregulated in the heart tissue of infants with TOF. Furthermore, we went on to show, using the zebrafish model, that altering the expression of these same scaRNAs led to faulty mRNA processing and heart defects in the developing embryo. This review will examine how scaRNAs may influence spliceosome fidelity in exon retention during heart development and thus contribute to regulation of heart development.

Kinesin-14 molecular motors represent an essential class of proteins that bind microtubules and walk toward their minus-ends. Previous studies have described important roles for Kinesin-14 motors at microtubule minus-ends, but their role in regulating plus-end dynamics remains controversial. Kinesin-14 motors have been shown to bind the EB family of microtubule plus-end binding proteins, suggesting that these minus-end–directed motors could interact with growing microtubule plus-ends. In this work, we explored the role of minus-end–directed Kinesin-14 motor forces in controlling plus-end microtubule dynamics. In cells, a Kinesin-14 mutant with reduced affinity to EB proteins led to increased microtubule lengths. Cell-free biophysical microscopy assays were performed using Kinesin-14 motors and an EB family marker of growing microtubule plus-ends, Mal3, which revealed that when Kinesin-14 motors bound to Mal3 at growing microtubule plus-ends, the motors subsequently walked toward the minus-end, and Mal3 was pulled away from the growing microtubule tip. Strikingly, these interactions resulted in an approximately twofold decrease in the expected postinteraction microtubule lifetime. Furthermore, generic minus-end–directed tension forces, generated by tethering growing plus-ends to the coverslip using λ-DNA, led to an approximately sevenfold decrease in the expected postinteraction microtubule growth length. In contrast, the inhibition of Kinesin-14 minus-end–directed motility led to extended tip interactions and to an increase in the expected postinteraction microtubule lifetime, indicating that plus-ends were stabilized by nonmotile Kinesin-14 motors. Together, we find that Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate microtubule lengths in cells.

Neuroscience researchers that wish to address compelling questions in psychopharmacology would benefit from the use of zebrafish, which have a behavioral repertoire that is rich and complex and that reflects many fundamental processes in humans, such as those that evoke anxiety. Zebrafish behavior is easily quantified in diverse test environments, but it is important to consider the conditions used to house the fish, prior to the behavioral testing, when designing experiments. Studies show that fish housed at high densities exhibit elevated cortisol levels as compared with fish housed at lower densities. The literature is less clear about the behavioral consequences of housing and handling. We examined the effects of housing (group versus paired) on several behavioral parameters. We observed that group-housed fish exhibited an anxious phenotype as evidenced by tank diving and meandering. Other more general indicators of movement and spatial navigation were unaffected by housing. This study focused on two specific housing environments, and the results support the need for continued research in this area to further elucidate the contributions of housing and handling on the subtleties of zebrafish behavioral markers.

Understanding the regulation of development can help elucidate the pathogenesis behind many developmental defects found in humans and other vertebrates. Evidence has shown that alternative splicing of messenger RNA (mRNA) plays a role in developmental regulation, but our knowledge of the underlying mechanisms that regulate alternative splicing are incomplete. Notably, a subset of small noncoding RNAs known as scaRNAs (small cajal body associated RNAs) contribute to spliceosome maturation and function through guiding covalent modification of spliceosomal RNAs with either methylation or pseudouridylation on specific nucleotides, but the developmental significance of these modifications is not well understood. Our focus is on one such scaRNA, known as SNORD94 or U94, that guides methylation on one specific cytosine (C62) on spliceosomal RNA U6, thus potentially altering spliceosome function during embryogenesis. We previously showed that in the myocardium of infants with heart defects, mRNA is alternatively spliced as compared to control tissues. We also demonstrated that alternatively spliced genes were concentrated in the pathways that control heart development. Furthermore, we showed that modifying expression of scaRNAs alters mRNA splicing in human cells, and zebrafish embryos. Here we present evidence that SNORD94 levels directly influence levels of methylation at its target region in U6, suggesting a potential mechanism for modifying alternative splicing of mRNA. The potential importance of scaRNAs as a developmentally important regulatory mechanism controlling alternative splicing of mRNA is unappreciated and needs more research.

DCB) 9 3 31under investigation. Alternative splicing of mRNA transcripts of genes is tissue and time specific 32 throughout life, although this process occurs everywhere in the body according to local tissue 33 needs and signals. The spliceosome, the large ribonucleoprotein complex that carries out splicing, 34 is biochemically modified by small noncoding RNAs, which is important for its structure and 35 function. Here we show that the amount of 2'-O-ribose methylation at nucleotide C 62 in 36 spliceosomal RNA U6 is dependent on the level of the scaRNA SNORD94. We hypothesize that 37 alternative splicing is dependent, at least in part, on biochemical modification to the spliceosomal 38 RNAs. Furthermore, when scaRNA directed modifications are dysregulated, the result causes 39 inappropriate alternative splicing that may contribute to developmental defects such as 40 congenital heart defects. To our knowledge, this is the first demonstration that 2'-O-ribose 41 methylation is indeed dependent on scaRNA levels in human cells and tissues. 42