Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate dynamic instability.

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.

Snord94 expression level alters methylation at C62 in snRNA U6.

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.

The Role of scaRNAs in Adjusting Alternative mRNA Splicing in Heart Development.

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.