Nucleolar detention of NONO shields DNA double-strand breaks from aberrant transcripts.

RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.

Mapping of a major QTL for increased robustness and detection of genome assembly errors in Asian seabass (Lates calcarifer).

BACKGROUND: For Asian seabass (Lates calcarifer, Bloch 1790) cultured at sea cages various aquatic pathogens, complex environmental and stress factors are considered as leading causes of disease, causing tens of millions of dollars of annual economic losses. Over the years, we conducted farm-based challenges by exposing Asian seabass juveniles to complex natural environmental conditions. In one of these challenges, we collected a total of 1,250 fish classified as either 'sensitive' or 'robust' individuals during the 28-day observation period. RESULTS: We constructed a high-resolution linkage map with 3,089 SNPs for Asian seabass using the double digest Restriction-site Associated DNA (ddRAD) technology and a performed a search for Quantitative Trait Loci (QTL) associated with robustness. The search detected a major genome-wide significant QTL for increased robustness in pathogen-infected marine environment on linkage group 11 (ASB_LG11; 88.9 cM to 93.6 cM) with phenotypic variation explained of 81.0%. The QTL was positioned within a > 800 kb genomic region located at the tip of chromosome ASB_LG11 with two Single Nucleotide Polymorphism markers, R1-38468 and R1-61252, located near to the two ends of the QTL. When the R1-61252 marker was validated experimentally in a different mass cross population, it showed a statistically significant association with increased robustness. The majority of thirty-six potential candidate genes located within the QTL have known functions related to innate immunity, stress response or disease. By utilizing this ddRAD-based map, we detected five mis-assemblies corresponding to four chromosomes, namely ASB_LG8, ASB_LG9, ASB_LG15 and ASB_LG20, in the current Asian seabass reference genome assembly. CONCLUSION: According to our knowledge, the QTL associated with increased robustness is the first such finding from a tropical fish species. Depending on further validation in other stocks and populations, it might be potentially useful for selecting robust Asian seabass lines in selection programs.

The glycolytic enzyme ALDOA and the exon junction complex protein RBM8A are regulators of ribosomal biogenesis.

Cellular growth is a fundamental process of life and must be precisely controlled in multicellular organisms. Growth is crucially controlled by the number of functional ribosomes available in cells. The production of new ribosomes depends critically on the activity of RNA polymerase (RNAP) II in addition to the activity of RNAP I and III, which produce ribosomal RNAs. Indeed, the expression of both, ribosomal proteins and proteins required for ribosome assembly (ribosomal biogenesis factors), is considered rate-limiting for ribosome synthesis. Here, we used genetic screening to identify novel transcriptional regulators of cell growth genes by fusing promoters from a ribosomal protein gene (Rpl18) and from a ribosomal biogenesis factor (Fbl) with fluorescent protein genes (RFP, GFP) as reporters. Subsequently, both reporters were stably integrated into immortalized mouse fibroblasts, which were then transduced with a genome-wide sgRNA-CRISPR knockout library. Subsequently, cells with altered reporter activity were isolated by FACS and the causative sgRNAs were identified. Interestingly, we identified two novel regulators of growth genes. Firstly, the exon junction complex protein RBM8A controls transcript levels of the intronless reporters used here. By acute depletion of RBM8A protein using the auxin degron system combined with the genome-wide analysis of nascent transcription, we showed that RBM8A is an important global regulator of ribosomal protein transcripts. Secondly, we unexpectedly observed that the glycolytic enzyme aldolase A (ALDOA) regulates the expression of ribosomal biogenesis factors. Consistent with published observations that a fraction of this protein is located in the nucleus, this may be a mechanism linking transcription of growth genes to metabolic processes and possibly to metabolite availability.

Tumor-Derived Lactic Acid Modulates Activation and Metabolic Status of Draining Lymph Node Stroma.

Communication between tumors and the stroma of tumor-draining lymph nodes (TDLN) exists before metastasis arises, altering the structure and function of the TDLN niche. Transcriptional profiling of fibroblastic reticular cells (FRC), the dominant stromal population of lymph nodes, has revealed that FRCs in TDLNs are reprogrammed. However, the tumor-derived factors driving the changes in FRCs remain to be identified. Taking an unbiased approach, we have shown herein that lactic acid (LA), a metabolite released by cancer cells, was not only secreted by B16.F10 and 4T1 tumors in high amounts, but also that it was enriched in TDLNs. LA supported an upregulation of Podoplanin (Pdpn) and Thy1 and downregulation of IL7 in FRCs of TDLNs, making them akin to activated fibroblasts found at the primary tumor site. Furthermore, we found that tumor-derived LA altered mitochondrial function of FRCs in TDLNs. Thus, our results demonstrate a mechanism by which a tumor-derived metabolite connected with a low pH environment modulates the function of fibroblasts in TDLNs. How lymph node function is perturbed to support cancer metastases remains unclear. The authors show that tumor-derived LA drains to lymph nodes where it modulates the function of lymph node stromal cells, prior to metastatic colonization.

Targeted protein degradation reveals a direct role of SPT6 in RNAPII elongation and termination.

SPT6 is a histone chaperone that tightly binds RNA polymerase II (RNAPII) during transcription elongation. However, its primary role in transcription is uncertain. We used targeted protein degradation to rapidly deplete SPT6 in human cells and analyzed defects in RNAPII behavior by a multi-omics approach and mathematical modeling. Our data indicate that SPT6 is a crucial factor for RNAPII processivity and is therefore required for the productive transcription of protein-coding genes. Unexpectedly, SPT6 also has a vital role in RNAPII termination, as acute depletion induced readthrough transcription for thousands of genes. Long-term depletion of SPT6 induced cryptic intragenic transcription, as observed earlier in yeast. However, this phenotype was not observed upon acute SPT6 depletion and therefore can be attributed to accumulated epigenetic perturbations in the prolonged absence of SPT6. In conclusion, targeted degradation of SPT6 allowed the temporal discrimination of its function as an epigenetic safeguard and RNAPII elongation factor.

PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A kinase.

The mitotic kinase AURORA-A is essential for cell cycle progression and is considered a priority cancer target. Although the catalytic activity of AURORA-A is essential for its mitotic function, recent reports indicate an additional non-catalytic function, which is difficult to target by conventional small molecules. We therefore developed a series of chemical degraders (PROTACs) by connecting a clinical kinase inhibitor of AURORA-A to E3 ligase-binding molecules (for example, thalidomide). One degrader induced rapid, durable and highly specific degradation of AURORA-A. In addition, we found that the degrader complex was stabilized by cooperative binding between AURORA-A and CEREBLON. Degrader-mediated AURORA-A depletion caused an S-phase defect, which is not the cell cycle effect observed upon kinase inhibition, supporting an important non-catalytic function of AURORA-A during DNA replication. AURORA-A degradation induced rampant apoptosis in cancer cell lines and thus represents a versatile starting point for developing new therapeutics to counter AURORA-A function in cancer.

MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation.

The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.

BAC-pool sequencing and analysis confirms growth-associated QTLs in the Asian seabass genome.

The Asian seabass is an important marine food fish that has been cultured for several decades in Asia Pacific. However, the lack of a high quality reference genome has hampered efforts to improve its selective breeding. A 3D BAC pool set generated in this study was screened using 22 SSR markers located on linkage group 2 which contains a growth-related QTL region. Seventy-two clones corresponding to 22 FPC contigs were sequenced by Illumina MiSeq technology. We co-assembled the MiSeq-derived scaffolds from each FPC contig with error-corrected PacBio reads, resulting in 187 sequences covering 9.7 Mb. Eleven genes annotated within this region were found to be potentially associated with growth and their tissue-specific expression was investigated. Correlation analysis demonstrated that SNPs in ctsb, skp1 and ppp2ca can be potentially used as markers for selecting fast-growing fingerlings. Conserved syntenies between seabass LG2 and five other teleosts were identified. This study i) provided a 10 Mb targeted genome assembly; ii) demonstrated NGS of BAC pools as a potential approach for mining candidates underlying QTLs of this species; iii) detected eleven genes potentially responsible for growth in the QTL region; and iv) identified useful SNP markers for selective breeding programs of Asian seabass.