SUNDAY, DECEMBER 4- Late Abstracts 1 - Molecular Biology of the ...

SUNDAY
We set out to identify the determinants of SUN-KASH interaction and to understand complex
formation in molecular detail. Using pulldown experiments, we were able to reconstitute SUN-
KASH binding in vitro. We analyzed luminal constructs of human SUN1 and SUN2 expressed
either in the ER of Hela cells or purified from E. coli. Both the conserved SUN domains and the
preceding coiled-coil regions were required for interaction. Replacing the endogenous coiledcoils
of SUN1 or SUN2 by an unrelated coiled-coil domain allowed efficient KASH binding. This
indicates that oligomerization of SUN domains by coiled-coils is a key determinant of SUN-
KASH interaction.
The crystal structure of the complex between the SUN domain of human SUN2 and the
Nesprin-2 KASH domain (see poster presented by Brian Sosa and Thomas Schwartz) revealed
essential features of SUN-KASH interaction in molecular detail. Using mutational analyses of
both binding partners, we confirmed the key determinants in in vitro binding assays.
Furthermore, we could show that the disruption of SUN-KASH interaction affects nuclear
envelope targeting of both SUN and KASH proteins.
2022
NSrp70 is a novel nuclear speckle-related protein and modulates alternative pre-mRNA
splicing in vivo.
Y-D. Kim 1 , J-Y. Lee 1 , K-M. Oh 1 ; 1 Life Science, Gwangju Inst Science/Technol, Gwangju, Korea
Nuclear speckle is known to serve as the storing place of mRNA splicing regulators. We report
here the identification and characterization of a novel speckle protein, referred to as NSrp70,
based on its sub-cellular location and apparent molecular weight. This protein was firstly
identified as the NSrp70, as the product of the NIH Mammalian Gene Collection, while no
function has been assigned yet. NSrp70 was co-localized and physically interacted with both
SC35 and ASF/SF2 in speckles. NSrp66 has putative RNA recognition motif, RE/RD rich
domain, and two coiled-coil domains, suggesting role in RNA processing. Accordingly, using
CD44, Tra2b1 and Fas constructs as splicing reporter minigenes, we found that NSrp70
modulated alternative splice site selection in vivo. The C-terminal ten amino acids (aa) (531-
540) including 536 RD 537 were identified as the novel nuclear localization signal, and the region
spanning 290 to 471 aa was critical for the speckle localization and the binding to SC35 and
ASF/SF2. The N-terminal region (107-161) was essential for the pre-mRNA splicing activity.
Finally, we found that knockout of NSrp70 gene in mice produced no progeny including fetal
embryos. Collectively, we demonstrate that the NSrp70 is a novel splicing regulator and
essentially required earlier during embryonic development.
2023
Quality control and stress response in mammalian ribosomal biogenesis.
M. Wang 1,2 , D. Pestov 2 ; 1 UMDNJ-GSBS, Stratford, NJ, 2 Cell Biology, UMDNJ-SOM, Stratford,
NJ
Ribosome is the subcellular component that translates mRNA to proteins. This complex
molecular machine is composed of two subunits, and contains four different ribosomal RNAs
(rRNAs) and 79 ribosomal proteins (RPs). The assembly of eukaryotic ribosomes is a multi-step
process involving numerous of proteins and RNA factors. Compromised ribosome integrity
contributes to premature aging syndromes, cancer and other diseases. Surveillance
mechanisms act throughout the entire ribosome synthesis pathway to eliminate defective
intermediates and thereby promote structural and functional integrity of the final ribosomal
particles. However, ribosome biogenesis is very sensitive to various disturbances of cellular