Spermatogenesis requires a large number of proteins to be properly expressed at certain stages, during which post-transcriptional regulation plays an important role. RNA-binding proteins (RBPs) are key players in post-transcriptional regulation, but only a few RBPs have been recognized and preliminary explored their function in spermatogenesis at present. Here we identified a new RBP tubby-like protein 2 (TULP2) and found three potential deleterious missense mutations of Tulp2 gene in dyszoospermia patients. Therefore, we explored the function and mechanism of TULP2 in male reproduction. TULP2 was specifically expressed in the testis and localized to spermatids. Studies on Tulp2 knockout mice demonstrated that the loss of TULP2 led to male sterility; on the one hand, increases in elongated spermatid apoptosis and restricted spermatid release resulted in a decreased sperm count; on the other hand, the abnormal differentiation of spermatids induced defective sperm tail structures and reduced ATP contents, influencing sperm motility. Transcriptome sequencing of mouse testis revealed the potential target molecular network of TULP2, which played its role in spermatogenesis by regulating specific transcripts related to the cytoskeleton, apoptosis, RNA metabolism and biosynthesis, and energy metabolism. We also explored the potential regulator of TULP2 protein function by using immunoprecipitation and mass spectrometry analysis, indicating that TUPL2 might be recognized by CCT8 and correctly folded by the CCT complex to play a role in spermiogenesis. Our results demonstrated the important role of TULP2 in spermatid differentiation and male fertility, which could provide an effective target for the clinical diagnosis and treatment of patients with oligo-astheno-teratozoospermia, and enrich the biological theory of the role of RBPs in male reproduction.

Tubby-like proteins (TULPs) are characterized by a conserved C-terminal domain that binds phosphoinositides. Collectively, mammalian TULP1-4 proteins play essential roles in intracellular transport, cell differentiation, signaling, and motility. Yet, little is known about how the function of these proteins is regulated in cells. Here, we present the protein-protein interaction network of TULP3, a protein that is responsible for the trafficking of G-protein-coupled receptors to cilia and whose aberrant expression is associated with severe developmental disorders and polycystic kidney disease. We identify several protein interaction nodes linked to TULP3 that include enzymes involved in acetylation and ubiquitination. We show that acetylation of two key lysine residues on TULP3 by p300 increases TULP3 protein abundance and that deacetylation of these sites by HDAC1 decreases protein levels. Furthermore, we show that one of these sites is ubiquitinated in the absence of acetylation and that acetylation inversely correlates with ubiquitination of TULP3. This mechanism is evidently conserved across species and is active in zebrafish during development. Finally, we identify this same regulatory module in TULP1, TULP2, and TULP4 and demonstrate that the stability of these proteins is similarly modulated by an acetylation switch. This study unveils a signaling pathway that links nuclear enzymes to ciliary membrane receptors via TULP3, describes a dynamic mechanism for the regulation of all tubby-like proteins, and explores how to exploit it pharmacologically using drugs.

Lung adenocarcinoma (LUAD) is one of the most common cancers and lethal diseases in the world. Recognition of the undetermined lung nodules at an early stage is useful for a favorable prognosis. However, there is no good method to identify the undetermined lung nodules and predict their clinical outcome. DNA methylation alteration is frequently observed in LUAD and may play important roles in carcinogenesis, diagnosis, and prediction. This study took advantage of publicly available methylation profiling resources and a machine learning method to investigate methylation differences between LUAD and adjacent non-malignant tissue. The prediction panel was first constructed using 338 tissue samples from LUAD patients including 149 non-malignant ones. This model was then validated with data from The Cancer Genome Atlas database and clinic samples. As a result, the methylation status of four CpG loci in homeobox A9 (HOXA9), keratin-associated protein 8-1 (KRTAP8-1), cyclin D1 (CCND1), and tubby-like protein 2 (TULP2) were highlighted as informative markers. A random forest classification model with an accuracy of 94.57% and kappa of 88.96% was obtained. To evaluate this panel for LUAD, the methylation levels of four CpG loci in HOXA9, KRTAP8-1, CCND1, and TULP2 of tumor samples and matched adjacent lung samples from 25 patients with LUAD were tested. In these LUAD patients, the methylation of HOXA9 was significantly upregulated, whereas the methylation of KRTAP8-1, CCND1, and TULP2 were downregulated obviously in tumor samples compared with adjacent tissues. Our study demonstrates that the methylation of HOXA9, KRTAP8-1, CCND1, and TULP2 has great potential for the early recognition of LUAD in the undetermined lung nodules. The findings also exhibit that the application of improved mathematic algorithms can yield accurate and particularly robust and widely applicable marker panels. This approach could greatly facilitate the discovery process of biomarkers in various fields.