Nonsense-mediated mRNA decay uses 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. 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 eightfold 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.

TP53 Null AML Line Does Not Selectively Expand Under Lenalidomide Treatment

Lenalidomide is an immunomodulatory therapeutic (IMiD) with known anti-proliferative and anti-angiogenic effects, and is an approved therapy for both 5q- Myelodysplastic Syndromes and Multiple Myeloma (MM). This thalidomide analog is known to retarget the E3 ubiquitin ligase cereblon to differential ubiquitination, and therefore degradation, of specific substrates. While lenalidomide maintenance is known to promote progression-free survival, increased rates of therapy-related myeloid neoplasms (TMNs) have been observed in MM patients on IMiD maintenance therapy following autologous stem cell transplant. Of note, TP53 is the most commonly mutated gene in TMN arising after lenalidomide therapy (Mouhieddine et al. 2020). TP53 is also recurrently mutated in TMNs following chemotherapy, suggesting that genotoxic stress results in the selective expansion of hematopoietic stem and progenitor cells (HSPCs) carrying mutations in TP53 (TN Wong et al. 2018). Based on these observations, we hypothesize that lenalidomide may contribute to the development of TMN by providing a fitness advantage to HSPCs carrying TP53 mutations. To test this hypothesis, we generated TP53 -/- deficient MOLM13 AML cells using CRISPR-Cas9 gene editing. We confirmed guide editing efficiency by next generation sequencing of a bulk edited pool of cells and generated isogenic clones via outgrowth of singly sorted cells, in an accepted approach for the field (Boettcher et al. 2019). We validated clones via sequencing and western blotting, and identified no differences in basal proliferation characteristics. Next, we further compared TP53 -/- deficient clones' with TP53 +/+ clones' response to cisplatin treatment (0.05-10µM) and observed a 2.8 fold increase in IC50 for TP53 -/- deficient clones. Satisfied that TP53 -/- isogenic clones successfully recapitulated this known resistance phenotype, we examined the response to lenalidomide by mixing TP53 -/- and TP53 +/+(AAVS1 edited control) clones and measuring expansion under lenalidomide treatment. However, we observed substantial inter-clone heterogeneity, with widely varying cellular proliferation characteristics following clone mixing, despite our prior validation. Therefore, we shifted to using bulk edited MOLM-13 cells in order to mitigate the potential impact of off-target effects and strong selective pressure of singly sorted clones. We edited a pool of cells at either TP53 or the safe harbor locus AAVS1 , allowed cells to recover, and mixed the two edited populations before treating with PBS, vehicle (DMSO), 1µm or 10µM lenalidomide for up to 28 days. We sequenced both AAVS1 and TP53 loci to measure cellular expansion by tracking the fraction of unedited, safe harbor edited, and TP53-/- genomic DNA at 0, 3, 7, and 28 days. Contrary to our hypothesis, TP53 -/- cells did not expand over time in response to lenalidomide treatment, and we did not detect a difference in TP53 -/- cell expansion with lenalidomide treatment compared to vehicle control (Figure 1). While we did not detect a cell intrinsic effect of TP53 mutations on lenalidomide response, it is possible that there are cell extrinsic effects, such as alterations in the bone marrow microenvironment, that contribute to the development of TP53-mutant TMN. It is also possible that a more prolonged exposure of lenalidomide is needed to see a significant effect of TP53-mutant cell expansion. This is relevant, since patients may be maintained on lenalidomide therapy for years. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Application Dilemma and Resolution Path of Safe Harbor Rules in the Field of Network Infringement

This paper mainly discusses the origin and content of the “safe harbor” rules of online trading platform providers in online infringement rules, and compares and analyzes the legislative consideration and development of the safe harbor principle in China with the provisions of the safe harbor principle in the United States. Through the legislative sorting of online infringement safe harbor rules, the paper explores the existing problems and legislative considerations of the specific application of the transplanted and introduced “safe harbor” rules in China’s e-commerce trading platform. By comparing and learning from foreign regulatory legislation, this paper combs out the applicability and path choice of “safe harbor” rule in China.

Identification of the safe harbor locus, AAVS1, from porcine genome and site-specific integration of recombinase-mediated cassette exchange system in porcine fibroblasts using CRISPR/Cas9

Gene integration at site-specific loci , such as safe harbor regions for s table expression via transgenesis , is a critical approach for understanding the function of a gene in cells or animals. The AAVS1 locus is a well-known safe harbor site for human and mouse studies. In the present study, we found an AAVS1-like sequence in the porcine genome using the UCSC Genome Browser and designed TALEN and CRISPR/Cas9 to target AAVS1. The efficiency of CRISPR/Cas9 for targeting the AAVS1 locus in porcine cells was superior to that of TALEN. An AAVS1-targeting donor vector containing GFP was designed and cloned. We added a loxP-lox2272 cassette sequence to the donor vector for further exchange of various transgenes in the AAVS1-targeted cell line. The donor vector and CRISPR/Cas9 components targeting AAVS1 were transfected into a porcine fibroblast cell line. Targeted cells of CRISPR/Cas9-mediated homologous recombination were identified by antibiotic selection. Gene knock-in at the AAVS1 locus was confirmed by PCR analysis. To induce recombinase-mediated cassette exchange (RMCE), another donor vector containing the loxP-lox2272 cassette and inducible (Tet-on) Cre recombinase was cloned. The Cre-donor vector was transfected into the AAVS1-targeted cell line, and RMCE was induced by adding doxycycline to the culture medium. RMCE in porcine fibroblasts was confirmed using PCR analysis. In conclusion, gene targeting at the AAVS1 locus and RMCE in porcine fibroblasts was successful. This technology will be useful for future porcine transgenesis studies and the generation of stable transgenic pigs.

PERFORMATIVE MEDIA POLICY: SECTION 230’S EVOLUTION FROM FOOTNOTE TO LOYALTY OATH

This study examines the legislative evolution of Section 230 of the Communications Act of 1934, a widely discussed and frequently misunderstood dimension of American telecommunications policy that provides a “safe harbor” provisionally shielding internet companies from liability for law-breaking content published by third parties who use their platforms and networks. Though this provision originated in the mid-1990s as an effort to minimize the legal and economic risks facing fledgling internet startups, we argue that efforts to reform it during the Trump era reflected an unprecedented transformation of an arcane policy point into a highly public subject for “messaging bills” intended principally to signal political loyalty to the president.