Ubiquitin-like protein 5 is a novel player in the UPR-PERK arm and ER stress-induced cell death.

Biological functions of the highly conserved ubiquitin-like protein 5 (UBL5) are not well understood. In Caenorhabditis elegans, UBL5 is induced under mitochondrial stress to mount the mitochondrial unfolded protein response (UPR). However, the role of UBL5 in the more prevalent endoplasmic reticulum (ER) stress-UPR in the mammalian system is unknown. In the present work, we demonstrated that UBL5 was an ER stress-responsive protein, undergoing rapid depletion in mammalian cells and livers of mice. The ER stress-induced UBL5 depletion was mediated by proteasome-dependent yet ubiquitin-independent proteolysis. Activation of the protein kinase R-like ER kinase arm of the UPR was essential and sufficient for inducing UBL5 degradation. RNA-Seq analysis of UBL5-regulated transcriptome revealed that multiple death pathways were activated in UBL5-silenced cells. In agreement with this, UBL5 knockdown induced severe apoptosis in culture and suppressed tumorigenicity of cancer cells in vivo. Furthermore, overexpression of UBL5 protected specifically against ER stress-induced apoptosis. These results identify UBL5 as a physiologically relevant survival regulator that is proteolytically depleted by the UPR-protein kinase R-like ER kinase pathway, linking ER stress to cell death.

UBL5/Hub1: An Atypical Ubiquitin-Like Protein with a Typical Role as a Stress-Responsive Regulator.

Members of the ubiquitin-like protein family are known for their ability to modify substrates by covalent conjugation. The highly conserved ubiquitin relative UBL5/Hub1, however, is atypical because it lacks a carboxy-terminal di-glycine motif required for conjugation, and the whole E1-E2-E3 enzyme cascade is likely absent. Though the conjugation-mediated role of UBL5/Hub1 is controversial, it undoubtedly functions by interacting non-covalently with its partners. Several interactors of UBL5/Hub1 identified to date have suggested broad stress-responsive functions of the protein, for example, stress-induced control of pre-mRNA splicing, Fanconi anemia pathway of DNA damage repair, and mitochondrial unfolded protein response. While having an atypical mode of function, UBL5/Hub1 is still a stress protein that regulates feedback to various stimuli in a similar manner to other ubiquitin-like proteins. In this review, I discuss recent progress in understanding the functions of UBL5/Hub1 and the fundamental questions which remain to be answered.

Ubiquitin-like proteins in the DNA damage response: the next generation.

DNA suffers constant insult from a variety of endogenous and exogenous sources. To deal with the arising lesions, cells have evolved complex and coordinated pathways, collectively termed the DNA damage response (DDR). Importantly, an improper DDR can lead to genome instability, premature ageing and human diseases, including cancer as well as neurodegenerative disorders. As a crucial process for cell survival, regulation of the DDR is multi-layered and includes several post-translational modifications. Since the discovery of ubiquitin in 1975 and the ubiquitylation cascade in the early 1980s, a number of ubiquitin-like proteins (UBLs) have been identified as post-translational modifiers. However, while the importance of ubiquitin and the UBLs SUMO and NEDD8 in DNA damage repair and signalling is well established, the roles of the remaining UBLs in the DDR are only starting to be uncovered. Herein, we revise the current status of the UBLs ISG15, UBL5, FAT10 and UFM1 as emerging co-regulators of DDR processes. In fact, it is becoming clear that these post-translational modifiers play important pleiotropic roles in DNA damage and/or associated stress-related cellular responses. Expanding our understanding of the molecular mechanisms underlying these emerging UBL functions will be fundamental for enhancing our knowledge of the DDR and potentially provide new therapeutic strategies for various human diseases including cancer.

Intron specificity in pre-mRNA splicing.

The occurrence of spliceosomal introns in eukaryotic genomes is highly diverse and ranges from few introns in an organism to multiple introns per gene. Introns vary with respect to their lengths, strengths of splicing signals, and position in resident genes. Higher intronic density and diversity in genetically complex organisms relies on increased efficiency and accuracy of spliceosomes for pre-mRNA splicing. Since intron diversity is critical for functions in RNA stability, regulation of gene expression and alternative splicing, RNA-binding proteins, spliceosomal regulatory factors and post-translational modifications of splicing factors ought to make the splicing process intron-specific. We recently reported function and regulation of a ubiquitin fold harboring splicing regulator, Sde2, which following activation by ubiquitin-specific proteases facilitates excision of selected introns from a subset of multi-intronic genes in Schizosaccharomyces pombe (Thakran et al. EMBO J, https://doi.org/10.15252/embj.201796751 , 2017). By reviewing our findings with understandings of intron functions and regulated splicing processes, we propose possible functions and mechanism of intron-specific pre-mRNA splicing and suggest that this process is crucial to highlight importance of introns in eukaryotic genomes.

Error-Prone Splicing Controlled by the Ubiquitin Relative Hub1.

Accurate pre-mRNA splicing is needed for correct gene expression and relies on faithful splice site recognition. Here, we show that the ubiquitin-like protein Hub1 binds to the DEAD-box helicase Prp5, a key regulator of early spliceosome assembly, and stimulates its ATPase activity thereby enhancing splicing and relaxing fidelity. High Hub1 levels enhance splicing efficiency but also cause missplicing by tolerating suboptimal splice sites and branchpoint sequences. Notably, Prp5 itself is regulated by a Hub1-dependent negative feedback loop. Since Hub1-mediated splicing activation induces cryptic splicing of Prp5, it also represses Prp5 protein levels and thus curbs excessive missplicing. Our findings indicate that Hub1 mediates enhanced, but error-prone splicing, a mechanism that is tightly controlled by a feedback loop of PRP5 cryptic splicing activation.

Ubiquitin-like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway.

Ubiquitin and ubiquitin-like proteins (UBLs) function in a wide array of cellular processes. UBL5 is an atypical UBL that does not form covalent conjugates with cellular proteins and which has a known role in modulating pre-mRNA splicing. Here, we report an unexpected involvement of human UBL5 in promoting the function of the Fanconi anemia (FA) pathway for repair of DNA interstrand crosslinks (ICLs), mediated by a specific interaction with the central FA pathway component FANCI. UBL5-deficient cells display spliceosome-independent reduction of FANCI protein stability, defective FANCI function in response to DNA damage and hypersensitivity to ICLs. By mapping the sequence determinants underlying UBL5-FANCI binding, we generated separation-of-function mutants to demonstrate that key aspects of FA pathway function, including FANCI-FANCD2 heterodimerization, FANCD2 and FANCI monoubiquitylation and maintenance of chromosome stability after ICLs, are compromised when the UBL5-FANCI interaction is selectively inhibited by mutations in either protein. Together, our findings establish UBL5 as a factor that promotes the functionality of the FA DNA repair pathway.

UBL5 is essential for pre-mRNA splicing and sister chromatid cohesion in human cells.

UBL5 is an atypical ubiquitin-like protein, whose function in metazoans remains largely unexplored. We show that UBL5 is required for sister chromatid cohesion maintenance in human cells. UBL5 primarily associates with spliceosomal proteins, and UBL5 depletion decreases pre-mRNA splicing efficiency, leading to globally enhanced intron retention. Defective sister chromatid cohesion is a general consequence of dysfunctional pre-mRNA splicing, resulting from the selective downregulation of the cohesion protection factor Sororin. As the UBL5 yeast orthologue, Hub1, also promotes spliceosome functions, our results show that UBL5 plays an evolutionary conserved role in pre-mRNA splicing, the integrity of which is essential for the fidelity of chromosome segregation.