Search bioRxiv⌕ Search

Biology subjects

Welty, R.

Publications and source records attributed to Welty, R..

2 recordsLinked to original sources

Zα and Zβ domains of ADAR1 and ZBP1 bind to G-quadruplexes with low micromolar affinity

1While it is well established that the Z domains of ADAR1 and ZBP1 proteins bind Z-form-prone nucleic acids (Z-NAs), it has also been shown that the Z domain of ADAR1 binds DNA G-Quadruplexes (GQ). However, no binding partner of the structurally homologous Z{beta} domain of ADAR1 has been identified to date. Based on AlphaFold and molecular dynamics simulations, it has recently been suggested that the Z{beta} domain of ADAR1 targets its substrate by recognizing GQs. Here, we provide the first experimental evidence for Z{beta} domain binding to select G-quadruplex RNA and DNA in vitro, with structural specificity and low micromolar affinity. We also demonstrate that the Z domains of ZBP1 bind to both DNA and RNA GQs with similar affinity. These findings extend the range of potential functional roles for these proteins and open new hypotheses for testing in cells.

biophysics↗

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast

The recognition of 5 splice site (5 ss) is one of the earliest steps of pre-mRNA splicing. To better understand the mechanism and regulation of 5 ss recognition, we selectively humanized components of the yeast U1 snRNP to reveal the function of these components in 5 ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yeast U1 (yU1) snRNA and the 5 ss RNA duplex. We replaced the Zinc-Finger (ZnF) domain of yU1C with its human counterpart, which resulted in cold-sensitive growth phenotype and moderate splicing defects. Next, we added an auxin-inducible degron to yLuc7 protein and found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of PRP40 and Snu71 (two other essential yeast U1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yeast U1 snRNP, and splicing plays a major role in the regulation of mitochondria function in yeast.

biochemistry↗