Loss of Prm1 leads to defective chromatin protamination, impaired PRM2 processing, reduced sperm motility and subfertility in male mice

One of the key events during spermiogenesis is the hypercondensation of chromatin by substitution of the majority of histones by protamines. In humans and mice, protamine 1 (PRM1/Prm1) and protamine 2 (PRM2/Prm2), are expressed in a species-specific ratio. Using CRISPR-Cas9-mediated gene editing we generated Prm1-deficient mice and demonstrate, that Prm1+/- mice are subfertile while Prm1-/- are infertile. Prm1-deficiency was associated with higher levels of 8-OHdG, an indicator for reactive oxygen mediated DNA-damage. While Prm1+/- males displayed moderate increased levels of 8-OHdG virtually all sperm of Prm1-/- males displayed ROS mediated DNA damage. Consequently, DNA integrity was slightly hampered in Prm1+/-, while DNA was completely fragmented in Prm1-/- animals. Interestingly CMA3 staining which indicates protamine-free DNA revealed, that Prm1+/- sperm displayed high levels (93%), compared to Prm2+/- (29%) and WT (2%) sperm. This is not due to increased histone retention as demonstrated by mass spectrometry (MassSpec) of nuclear proteins in Prm1+/- sperm. Further analysis of the MassSpec data from sperm nuclear proteome revealed, that only one protein (RPL31) is significantly higher abundant in Prm1+/- compared to WT sperm. Comparison of the proteome from Prm1-/- and Prm2-/- to WT suggested, that there are a small number of proteins which differ in abundance. However, their function was not linked mechanistically to primary defects seen in Prm1-/- mice and rather represent a general stress response. Interestingly, using acid urea gels we found that sperm from Prm1+/- and Prm1-/- mice contain a high level of unprocessed, full-length PRM2. Prm2 is transcribed as a precursor protein which, upon binding to DNA is successively processed. Further, the overall ratio of PRM1:PRM2 is skewed from 1:2 in WT to 1:5 in Prm1+/- animals. Our results reveal that Prm1 is required for proper processing of PRM2 to produce the mature PRM2 which, together with Prm1 is able to hypercondense DNA. Hence, the species specific PRM1:PRM2 ratio has to be precisely controlled in order to retain full fertility.

Differential nuclear import sets the timing of protein access to the embryonic genome

The development of a fertilized egg to an embryo requires the proper temporal control of gene expression. During cell differentiation, timing is often controlled via cascades of transcription factors (TFs). However, in early development, transcription is often inactive, and many TF levels are constant, suggesting that unknown mechanisms govern the observed rapid and ordered onset of gene expression. Here, we find that in early embryonic development, access of maternally deposited nuclear proteins to the genome is temporally ordered via importin affinities, thereby timing the expression of downstream targets. We quantify changes in the nuclear proteome during early development and find that nuclear proteins, such as TFs and RNA polymerases, enter nuclei sequentially. Moreover, we find that the timing of the access of nuclear proteins to the genome corresponds to the timing of downstream gene activation. We show that the affinity of proteins to importin is a major determinant in the timing of protein entry into embryonic nuclei. Thus, we propose a mechanism by which embryos encode the timing of gene expression in early development via biochemical affinities. This process could be critical for embryos to organize themselves before deploying the regulatory cascades that control cell identities.

Neuronal activity regulates the nuclear proteome to promote activity-dependent transcription

The formation and plasticity of neuronal circuits relies on dynamic activity-dependent gene expression. Although recent work has revealed the identity of important transcriptional regulators and of genes that are transcribed and translated in response to activity, relatively little is known about the cell biological mechanisms by which activity alters the nuclear proteome of neurons to link neuronal stimulation to transcription. Using nucleus-specific proteomic mapping in silenced and stimulated neurons, we uncovered an understudied mechanism of nuclear proteome regulation: activity-dependent proteasome-mediated degradation. We found that the tumor suppressor protein PDCD4 undergoes rapid stimulus-induced degradation in the nucleus of neurons. We demonstrate that degradation of PDCD4 is required for normal activity-dependent transcription and that PDCD4 target genes include those encoding proteins critical for synapse formation, remodeling, and transmission. Our findings highlight the importance of the nuclear proteasome in regulating the activity-dependent nuclear proteome and point to a specific role for PDCD4 as a regulator of activity-dependent transcription in neurons.

Aging Induces Hepatic Oxidative Stress and Nuclear Proteomic Remodeling in Liver from Wistar Rats

Aging is a continuous, universal, and irreversible process that determines progressive loss of adaptability. The liver is a critical organ that supports digestion, metabolism, immunity, detoxification, vitamin storage, and hormone signaling. Nevertheless, the relationship between aging and the development of liver diseases remains elusive. In fact, although prolonged fasting in adult rodents and humans delays the onset of the disease and increases longevity, whether prolonged fasting could exert adverse effects in old organisms remains incompletely understood. In this work, we aimed to characterize the oxidative stress and nuclear proteome in the liver of 3-month- and 24-month-old male Wistar rats upon 36 h of fasting and its adaptation in response to 30 min of refeeding. To this end, we analyzed the hepatic lipid peroxidation levels (TBARS) and the expression levels of genes associated with fat metabolism and oxidative stress during aging. In addition, to gain a better insight into the molecular and cellular processes that characterize the liver of old rats, the hepatic nuclear proteome was also evaluated by isobaric tag quantitation (iTRAQ) mass spectrometry-based proteomics. In old rats, aging combined with prolonged fasting had great impact on lipid peroxidation in the liver that was associated with a marked downregulation of antioxidant genes (Sod2, Fmo3, and Cyp2C11) compared to young rats. Besides, our proteomic study revealed that RNA splicing is the hepatic nuclear biological process markedly affected by aging and this modification persists upon refeeding. Our results suggest that aged-induced changes in the nuclear proteome could affect processes associated with the adaptative response to refeeding after prolonged fasting, such as those involved in the defense against oxidative stress.

DYT-TOR1A Subcellular Proteomics Reveals Selective Vulnerability of the Nuclear Proteome to Cell Stress

TorsinA is a AAA+ ATPase that shuttles between the ER lumen and outer nuclear envelope in an ATP-dependent manner and is functionally implicated in nucleocytoplasmic transport. We hypothesized that the DYT-TOR1A dystonia disease-causing variant, ΔE TorsinA, may therefore disrupt the normal subcellular distribution of proteins between the nuclear and cytosolic compartments. To test this hypothesis, we performed proteomic analysis on nuclear and cytosolic subcellular fractions from DYT-TOR1A and wildtype mouse embryonic fibroblasts (MEFs). We further examined the compartmental proteomes following exposure to thapsigargin (Tg), an endoplasmic reticulum (ER) stressor, because DYT-TOR1A dystonia models have previously shown abnormalities in cellular stress responses. Across both subcellular compartments, proteomes of DYT-TOR1A cells showed basal state disruptions consistent with an activated stress response, and in response to thapsigargin, a blunted stress response. However, the DYT-TOR1A nuclear proteome under Tg cell stress showed the most pronounced and disproportionate degree of protein disruptions - 3-fold greater than all other conditions. The affected proteins extended beyond those typically associated with stress responses, including enrichments for processes critical for neuronal synaptic function. These findings highlight the advantage of subcellular proteomics to reveal events that localize to discrete subcellular compartments and refine thinking about the mechanisms and significance of cell stress in DYT-TOR1A pathogenesis.

LC-MS Based Draft Map of the Arabidopsis thaliana Nuclear Proteome and Protein Import in Pattern Triggered Immunity

Despite its central role as the ark of genetic information and gene expression the plant nucleus is surprisingly understudied. We isolated nuclei from the Arabidopsis thaliana dark grown cell culture left untreated and treated with flg22 and nlp20, two elicitors of pattern triggered immunity (PTI) in plants, respectively. An LC-MS based discovery proteomics approach was used to measure the nuclear proteome fractions. An enrichment score based on the relative abundance of cytoplasmic, mitochondrial and golgi markers in the nuclear protein fraction allowed us to curate the nuclear proteome producing high quality catalogs of around 3,000 nuclear proteins under untreated and both PTI conditions. The measurements also covered low abundant proteins including more than 100 transcription factors and transcriptional co-activators. Protein import into the nucleus in plant immunity is known. Here we sought to gain a broader impression of this phenomenon employing our proteomics data and found 157 and 73 proteins to be putatively imported into the nucleus upon stimulus with flg22 and nlp20, respectively. Furthermore, the abundance of 93 proteins changed significantly in the nucleus following elicitation of immunity. These results suggest promiscuous ribosome assembly and retrograde signaling from the mitochondrion to the nucleus including Prohibitins and Cytochrome C, in the two forms of PTI.

Neuronal activity promotes nuclear proteasome-mediated degradation of PDCD4 to regulate activity-dependent transcription

Activity-dependent gene expression is critical for synapse development and plasticity. To elucidate novel mechanisms linking neuronal activity to changes in transcription, we compared the nuclear proteomes of tetrodotoxin-silenced and bicuculline-stimulated cultured rodent neurons using nuclear-localized APEX2 proximity biotinylation and mass spectrometry. The tumor suppressor protein PDCD4 was enriched in the silenced nuclear proteome, and PDCD4 levels rapidly decreased in the nucleus and cytoplasm of stimulated neurons. The activity-dependent decrease of PDCD4 was prevented by inhibitors of both PKC and proteasome activity and by a phospho-incompetent mutation of Ser71 in the βTRCP ubiquitin ligase-binding motif of PDCD4. We compared the activity-dependent transcriptomes of neurons expressing wildtype or degradation-resistant (S71A) PDCD4. We identified 91 genes as PDCD4 targets at the transcriptional level, including genes encoding proteins critical for synapse formation, remodeling, and transmission. Our findings indicate that regulated degradation of nuclear PDCD4 facilitates activity-dependent transcription in neurons.

Analysis of rice nuclear-localized seed-expressed proteins and their database (RSNP-DB)

Nuclear proteins are primarily regulatory factors governing gene expression. Multiple factors determine the localization of a protein in the nucleus. An upright identification of nuclear proteins is way far from accuracy. We have attempted to combine information from subcellular prediction tools, experimental evidence, and nuclear proteome data to identify a reliable list of seed-expressed nuclear proteins in rice. Depending upon the number of prediction tools calling a protein nuclear, we could sort 19,441 seed expressed proteins into five categories. Of which, half of the seed-expressed proteins were called nuclear by at least one out of four prediction tools. Further, gene ontology (GO) enrichment and transcription factor composition analysis showed that 6116 seed-expressed proteins could be called nuclear with a greater assertion. Localization evidence from experimental data was available for 1360 proteins. Their analysis showed that a 92.04% accuracy of a nuclear call is valid for proteins predicted nuclear by at least three tools. Distribution of nuclear localization signals and nuclear export signals showed that the majority of category four members were nuclear resident proteins, whereas other categories have a low fraction of nuclear resident proteins and significantly higher constitution of shuttling proteins. We compiled all the above information for the seed-expressed genes in the form of a searchable database named Rice Seed Nuclear Protein DataBase (RSNP-DB) https://pmb.du.ac.in/rsnpdb. This information will be useful for comprehending the role of seed nuclear proteome in rice.