Characterization of biological functions of human RNA-binding proteins in Poly(ADP-ribose) polymerase-1-regulated pathways

Poly(ADP-ribosy)lation (PARylation) can served as a scaffold for noncovalent interactions with various RNA binding proteins including helicases and ribosomal proteins, but many important questions remains regarding the molecular functions and biological roles through the interaction. Based on live-cell imaging assay, I found that DEAD-box helicase 18 (DDX18) and ribosomal protein S19 (RPS19) accumulated to DNA damage sites via PARP-1 activation, indicating their participation in PARP-1-dependent DNA damage repair. My results show that PARP-1 can mediate the association of DDX18 with R-loops thereby modulating R-loop homeostasis and R-loop-dependent DNA damage. DDX18 knockdown renders cells more sensitive to DNA damaging reagents. Furthermore, knockdown of DDX18 reduces R-loop-induced RPA32 and RAD51 foci formation in response to irradiation, and DDX18 depletion also leads to R-loop-induced γH2AX accumulation and genome instability. In addition, DDX18 knockdown abolishes DNA replication due to R-loop accumulation. Taken together, the data uncover new functions of PARP-1 mediated DDX18 in R-loop-mediated events. Here, I also identify RPS19 as a new regulator in DNA double-strand break (DSB) repair. RPS19 was recruited to the DNA damage regions through its putative PAR-binding motif, and the accumulation of RPS19 was PARP-1 dependent. In the absence of RPS19, cells showed reduced RAD51 foci formation, whereas γH2AX and RPA32 foci were increased. Furthermore, the accumulation of RPS19 at DNA damage sites were abolished during the treatment of ATR inhibitors but not ATM inhibitors. Upon X-ray irradiation, RPS19-knockdown cells also decreased phosphorylation of Chk1 at Ser345, which is the downstream of ATR. The results suggest the novel role of RPS19 in ATR pathway during DNA damage repair. In summary, these observations provide a further mechanistic understanding of RNA-binding proteins in PARP-1 mediated DNA damage responses.Biochemistr

Quantitative Proteomics and Differential Protein Abundance Analysis after the Depletion of PEX3 from Human Cells Identifies Additional Aspects of Protein Targeting to the ER

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of around 10,000 different soluble and membrane proteins in humans. It involves the co- or post translational targeting of precursor polypeptides to the ER, and their subsequent membrane insertion or translocation. So far, three pathways for the ER targeting of precursor polypeptides and four pathways for the ER targeting of mRNAs have been described. Typically, these pathways deliver their substrates to the Sec61 polypeptide-conducting channel in the ER membrane. Next, the pre cursor polypeptides are inserted into the ER membrane or translocated into the ER lumen, which may involve auxiliary translocation components, such as the TRAP and Sec62/Sec63 complexes, or auxiliary membrane protein insertases, such as EMC and the TMCO1 complex. Recently, the PEX19/PEX3-dependent pathway, which has a well-known function in targeting and inserting vari ous peroxisomal membrane proteins into pre-existent peroxisomal membranes, was also found to act in the targeting and, putatively, insertion of monotopic hairpin proteins into the ER. These either remain in the ER as resident ER membrane proteins, or are pinched off from the ER as components of new lipid droplets. Therefore, the question arose as to whether this pathway may play a more general role in ER protein targeting, i.e., whether it represents a fourth pathway for the ER targeting of precursor polypeptides. Thus, we addressed the client spectrum of the PEX19/PEX3-dependent pathway in both PEX3-depleted HeLa cells and PEX3-deficient Zellweger patient fibroblasts by an established approach which involved the label-free quantitative mass spectrometry of the total proteome of depleted or deficient cells, as well as differential protein abundance analysis. The negatively affected proteins included twelve peroxisomal proteins and two hairpin proteins of the ER, thus confirming two previously identified classes of putative PEX19/PEX3 clients in human cells. Interestingly, fourteen collagen-related proteins with signal peptides or N-terminal transmembrane helices belonging to the secretory pathway were also negatively affected by PEX3 deficiency, which may suggest compromised collagen biogenesis as a hitherto-unknown contributor to organ failures in the respective Zellweger patients

Chemistry & Biochemistry Newsletter

Inside: Page 2 2003 Graduates; Bonneman Scholarship in Medical Technology Awarded; Dean’s List–Spring 2003 Page 3 Update of Sophomore Organic Chemistry Lab; Halaweish Receives Governor’s Grant Page 4 A Mini-sized Antarctic in Shepard Hall Page 5 Alumni News; Faculty and Staff News; Foundation Donors Page 6 Recent Faculty Publication

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Inside: Page 2 • Ph.D. in Biochemistry Approved for SDSU • Recent Faculty Publications Page 3 • Research Aims to Bring Back Traditional Food Page 4 • SDSU Chemistry Department Goes Green • The Twelve Principle of Green Chemistry Page 5 • New Graduate Students Page 6 • MLS Part of IHEP Documentary • MLS Student Sets New High on Board Exam Page 7 • New Faculty and Staff Page 8 • Recent Faculty Publication

Chemistry & Biochemistry Newsletter

Inside: Page 2 2003 Graduates; Bonneman Scholarship in Medical Technology Awarded; Dean’s List–Spring 2003 Page 3 Update of Sophomore Organic Chemistry Lab; Halaweish Receives Governor’s Grant Page 4 A Mini-sized Antarctic in Shepard Hall Page 5 Alumni News; Faculty and Staff News; Foundation Donors Page 6 Recent Faculty Publication

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Inside: Page 2 Photodynamic Therapy Offers New Approach to Cancer Page 3 Chemistry Professor Recognized by Peers as Member of the Year; Department Head Joins NSF Committee Page 4 Retired SDSU Dean Honored for Distance Education Alliance; Miller receives Edward Patrick Hogan Award; Freshman Chemistry Major Honored Page 5 2008 Chemistry and Biochemistry Graduates Page 6 Scholarships and Award

Chemistry & Biochemistry Newsletter

Inside: Page 2 • Ph.D. in Biochemistry Approved for SDSU • Recent Faculty Publications Page 3 • Research Aims to Bring Back Traditional Food Page 4 • SDSU Chemistry Department Goes Green • The Twelve Principle of Green Chemistry Page 5 • New Graduate Students Page 6 • MLS Part of IHEP Documentary • MLS Student Sets New High on Board Exam Page 7 • New Faculty and Staff Page 8 • Recent Faculty Publication

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Inside: Page 2 CLS: Rewarding Field Looks for Increased Awareness Page 3 Grant to Aid Ethanol Byproduct Research Page 4 Studying World History in a Cold, Cold Place Page 5 SDSU Research Finds Drugs React to Herbal Remedies; How’s Your Memory? Page 6 Alumni News; Faculty and Staff News; Scholarship and Award Winners; Dean’s List – Spring 2004 Page 7 New Grad Students; New Faculty Page 8 Exciting New Era for Development; Foundation Donor

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Inside: Page 2 New Graduate Students in Chemistry/Biochemistry Page 3 and 4 New Faculty Page 5 Alumni News; Faculty and Staff News; Foundation Donors Page 6 Recent Faculty Publication

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Inside: Page 2-3 BioSNTR Funding Boosts Biotechnology in South Dakota Page 3 New Student Club Decorates Tree Page 4-5 New Graduate Students Page 5 Arts & Sciences contributes 2,875 items to food pantry Page 6-7 New Faculty and Staff Page 7 Recent Faculty Publications Page 8 Student Engineers (and a biochemist) Without Border