Biology subjects

Udy, D. B.

Publications and source records attributed to Udy, D. B..

5 recordsLinked to original sources

bioRxiv2022

Recursive splicing discovery using lariats in total RNA sequencing

Recursive splicing is a noncanonical splicing mechanism in which an intron is removed in segments via multiple splicing reactions. Relatively few recursive splice sites have been identified with high confidence in human introns, and more comprehensive analyses are needed to better characterize where recursive splicing happens and whether or not it has a regulatory function. In this study, we use an unbiased approach utilizing intron lariats to search for recursive splice sites in constitutive introns and alternative exons in the human transcriptome. We find evidence for recursive splicing in a broader range of intron sizes than previously reported and detail a new location for recursive splicing at the distal ends of cassette exons. Additionally, we identify evidence for the conservation of these recursive splice sites among higher vertebrates and the use of these sites to influence alternative exon exclusion. Together, our data demonstrate the prevalence of recursive splicing and its potential influence on gene expression through alternatively spliced isoforms.

genomics↗

bioRxiv2021

Nonsense-mediated mRNA decay utilizes complementary mechanisms to suppress mRNA and protein accumulation

Nonsense-mediated mRNA decay (NMD) is an essential, highly conserved quality control pathway that detects and degrades mRNAs containing premature termination codons (PTCs). Although the essentiality of NMD is frequently ascribed to its prevention of truncated protein accumulation, the extent to which NMD actually suppresses proteins encoded by NMD-sensitive transcripts is less well-understood than NMD-mediated suppression of mRNA. Here, we describe a reporter system that permits accurate quantification of both mRNA and protein levels via stable integration of paired reporters encoding NMD-sensitive and NMD-insensitive transcripts into the AAVS1 safe harbor loci in human cells. We use this system to demonstrate that NMD suppresses proteins encoded by NMD-sensitive transcripts by up to [~]8-fold more than the mRNA itself. Our data indicate that NMD limits the accumulation of proteins encoded by NMD substrates by mechanisms beyond mRNA degradation, such that even when NMD-sensitive mRNAs escape destruction, their encoded proteins are still effectively suppressed.

molecular biology↗

bioRxiv2020

The origins and consequences of UPF1 variants in pancreatic adenosquamous carcinoma

Pancreatic adenosquamous carcinoma (PASC) is a rare and aggressive subtype of pancreatic cancer whose mutational origins are poorly understood. An early study reported somatic mutations in UPF1, which encodes a core component of the nonsense-mediated mRNA decay (NMD) pathway, as a common signature of PASC, but subsequent studies did not observe these lesions in other PASC cohorts. The corresponding controversy about whether UPF1 mutations are important contributors to PASC has been exacerbated by a paucity of functional studies of these lesions. Here, we systematically assessed the potential roles of UPF1 mutations in PASC. We modeled two reported UPF1 mutations to find no consistent effects on pancreatic cancer growth, acquisition of adenosquamous features, UPF1 splicing, UPF1 protein levels, or NMD efficiency. We subsequently discovered that ~40% of UPF1 mutations reportedly present in PASCs are identical to standing genetic variation in the human population, suggesting that they are likely non-pathogenic inherited variation rather than pathogenic mutations. Our data suggest that UPF1 is not a common functional driver of PASC and motivate further attempts to identify unique genetic features defining these malignancies.

cancer biology↗

bioRxiv2017

The mammalian kinetochore independently regulates its passive and active force-generating interfaces with microtubules

The kinetochore links chromosomes to dynamic spindle microtubules and drives both chromosome congression and segregation. To do so, the kinetochore must hold on to depolymerizing and polymerizing microtubules. At metaphase, one sister kinetochore couples to depolymerizing microtubules, pulling its sister along polymerizing microtubules [1,2]. Distinct kinetochore-microtubule interfaces mediate these behaviors: active interfaces transduce microtubule depolymerization into mechanical work, and passive interfaces generate friction as the kinetochore slides along microtubules [3,4]. We do not know the physical and molecular nature [5-7] of these interfaces, or how they are regulated to support diverse mitotic functions in mammalian cells. To address this question, we focus on the mechanical role of the essential load-bearing protein Hec1 [8-11]. Hec1s affinity for microtubules is regulated by Aurora B phosphorylation on its N-terminal tail [12-15], but its role at the passive and active interfaces remains unclear. Here, we use laser ablation to trigger cellular pulling on mutant kinetochores and decouple sisters in vivo, and thereby separately probe Hec1s role as it moves on polymerizing versus depolymerizing microtubules. We show that Hec1 phosphorylation tunes passive friction along polymerizing microtubules, modulating both the magnitude and timescale of responses to force. In contrast, we find that Hec1 phosphorylation does not affect the kinetochores ability to grip depolymerizing microtubules, or switch to this active force-generating state. Together, the data suggest that different kinetochore interfaces engage with growing and shrinking microtubules, and that passive friction can be regulated without disrupting active force generation. Through this mechanism, the kinetochore can modulate its grip on microtubules as its functional needs change during mitosis, and yet retain its ability to couple to microtubules powering chromosome movement.

cell biology↗

bioRxiv2017

Mapping load-bearing in the mammalian spindle reveals local kinetochore-fiber anchorage that provides mechanical isolation and redundancy

Active forces generated at kinetochores move chromosomes, and the dynamic spindle must robustly anchor kinetochore-fibers (k-fibers) to bear this load. We know that the mammalian spindle body can bear the load of chromosome movement far from poles, but do not know where and how - physically and molecularly - this load is distributed across the spindle. In part, this is because perturbing and reading out spindle mechanics in live cells is difficult. Yet, answering this question is key to understanding how the spindle generates and responds to force, and performs its diverse mechanical functions. Here, we map load-bearing across the mammalian spindle in space-time, and dissect local anchorage mechanics and mechanism. To do so, we laser ablate single k-fibers at different spindle locations, and in different molecular backgrounds, and quantify at high time resolution the immediate relaxation of chromosomes, k-fibers, and microtubule speckles. We find that load redistribution is locally confined in all directions: along the first 3-4 m from kinetochores, scaling with k-fiber length, and laterally within ~2 m of k-fiber sides, without neighboring k-fibers sharing load. A phenomenological model constrains the mechanistic underpinnings of these data: it suggests that dense, transient crosslinks to the spindle along k-fibers bear the load of chromosome movement, but that these connections do not limit the timescale of spindle reorganization. The microtubule crosslinker NuMA is needed for the local load-bearing observed, while Eg5 and PRC1 are not, suggesting specialization in mechanical function and a novel function for NuMA throughout the spindle body. Together, the data and model suggest that widespread NuMA-mediated crosslinks locally bear load, providing mechanical isolation and redundancy while allowing spindle fluidity. These features are well-suited to support robust chromosome segregation.

cell biology↗