Splicing factor SRSF1 tunes nucleolar cap pH to rescue ribosome factories after stress

Deep inside the cell nucleus, the nucleolus works around the clock as the factory where ribosomes, the protein-building machines of every living cell, are assembled. Far from being a shapeless blob, this structure is now understood as a multiphase condensate, a set of liquid-like compartments nested within one another and organized along an internal pH gradient. When cells are stressed, that delicate architecture can fall apart, and the question of how the nucleolus rebuilds itself has puzzled cell biologists for decades. A new study published in Nature Chemical Biology by Suibin Ma, Jierui Guo and colleagues in the laboratory of Bo Wang at Xiamen University reveals a surprising answer: a well-known RNA splicing factor called SRSF1 moonlights as a pH regulator, shuttling to a specific nucleolar compartment to restore the acidity balance needed for the organelle to recover its structure and function.

The nucleolus is organized into three subcompartments: the fibrillar centers, the dense fibrillar component and the granular component, each hosting a distinct stage of ribosomal RNA production and processing. Recent work has shown that these subcompartments are maintained as coexisting liquid phases, and that a pH gradient across them helps set up the biochemical conditions each phase requires. Yet while pH is recognized as a key variable controlling condensate dynamics and function, how cells actually control pH inside these compartments under physiological and pathological conditions has remained poorly understood. The Xiamen team set out to close that gap, focusing on a structure that appears when nucleolar transcription is disrupted.

When rRNA synthesis is interrogated by stress, whether from DNA damage or from drugs that block transcription, the nucleolus undergoes a characteristic reorganization. Some of its components segregate into cap-like structures at the nucleolar periphery, structures that were first described more than sixty years ago in cells treated with the chemotherapeutic agent actinomycin D. Among these are the so-called dark nucleolar caps, or DNCs, marked by proteins such as SFPQ and DDX18. The researchers discovered that under specific stressed conditions, SRSF1, a canonical splicing factor that normally resides in nuclear speckles, is recruited into these DNCs. Using super-resolution imaging and knock-in fluorescent tags in human cells, they tracked this redistribution in detail, finding that it occurs not only in one cell line but across multiple human cell types, including 293T, HCT 116 and Huh7 cells, after ultraviolet treatment followed by a recovery period.

Recruitment, however, is not random. Through immunoprecipitation combined with mass spectrometry, the team identified a molecular interaction between SRSF1 and DDX18, a DEAD-box RNA helicase that localizes to the granular component and the DNCs. When DDX18 was depleted with short hairpin RNA, SRSF1’s ability to accumulate in the dark nucleolar caps was impaired, indicating that this interaction is partially required for SRSF1’s stress-induced localization. The finding adds a new dimension to the biology of SR proteins, a family of splicing factors already known to redistribute to nuclear stress bodies and segregated nucleolar components in response to DNA damage, and it suggests that the nucleolar caps act as staging grounds where splicing machinery performs an unexpected, non-splicing job.

That job, the study shows, is pH management. Using live-cell imaging with the ratiometric pH-sensitive dye BCECF-AM and a genetically encoded pHluorin biosensor, the researchers measured the pH inside individual nucleolar subcompartments with remarkable precision. They found that the pH microenvironments of the granular component, the dense fibrillar component and the fibrillar centers remain largely unchanged upon ultraviolet exposure, but the dark nucleolar caps behave differently. When SRSF1 was knocked down, the pH homeostasis of the DNCs was disrupted, and this disturbance cascaded into a failure of the whole organelle to recover. Cells lacking SRSF1 could not properly restore the nucleolar multiphase architecture after stress, and functional readouts of nucleolar health deteriorated accordingly.

The functional consequences were striking. In control cells subjected to ultraviolet treatment or actinomycin D and then allowed to recover, the granular component protein NPM1 repartitioned back into the nucleolus, nascent RNA synthesis resumed as measured by 5-ethynyl uridine incorporation, and precursors of ribosomal RNA detected by probes against the 5-prime external transcribed spacer returned to normal levels. In SRSF1-depleted cells, all of these recovery markers were blunted. The team also observed elevated levels of gamma-H2AX, a marker of DNA damage, in the stressed cells lacking SRSF1, consistent with the idea that impaired nucleolar recovery leaves the genome more vulnerable. Rescue experiments in which shRNA-resistant SRSF1 was reintroduced restored DNC pH and nucleolar function, confirming the specificity of the effect.

The mechanistic heart of the paper lies in SRSF1’s arginine/serine-rich, or RS, domain. Because of its high positive charge, this domain is accountable for alkalizing the DNC microenvironment. When the researchers mutated the charged residues of the RS domain to uncharged alanines, the mutant SRSF1 failed to modulate the pH of the dark nucleolar caps even though it could still localize there. Importantly, the pH-modulating activity of SRSF1 was shown to operate independently of its canonical role in splicing regulation, separating this newly discovered function from the protein’s day job in RNA processing. The work resonates with a growing body of evidence that biomolecular condensates can sustain electrochemical gradients and modulate reactions at their interfaces, and with theoretical work suggesting that charge neutralization allows condensates to maintain pH gradients at equilibrium.

Perhaps the most translationally exciting result is the demonstration that the pH-restoring function can be mimicked by a synthetic molecule. The team designed arginine-rich dipeptide derivatives based on the SRSF1 RS domain, varying their length and net charge, and expressed them in stressed cells. These synthetic dipeptides localized to the nucleolus, enriched in the DNC region as measured against the DDX18 fluorescence mask, and safeguarded the nucleolus from pH and functional disturbance. In cells treated with actinomycin D and then allowed to recover, the arginine-arginine dipeptides preserved nucleolar size, maintained 5-prime ETS and rRNA levels, and protected the multiphase organization of the organelle. In other words, a minimal positively charged peptide was sufficient to stand in for the splicing factor’s pH-buffering role, offering a proof of principle that nucleolar pH can be pharmacologically tuned.

The implications extend well beyond basic cell biology. The nucleolus is increasingly viewed as a therapeutic target in human disease, and its disorganization is a hallmark of stress responses in cancer chemotherapy, since many chemotherapeutic drugs inhibit ribosome biogenesis at various levels. Nucleolar dysfunction has also been implicated in neurodegenerative conditions: dipeptide repeat proteins produced by the C9orf72 repeat expansion, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia, are known to disturb biomolecular phase separation and disrupt nucleolar function. A pathway that actively restores nucleolar pH homeostasis after stress, and that can be enhanced with simple arginine-rich peptides, suggests new angles for intervening in diseases where condensate chemistry goes wrong. It also reframes SRSF1, long studied as a splicing factor and oncoprotein, as a multifunctional regulator whose charged domains carry out electrochemical work inside membrane-less organelles.

Technically, the study stands out for the rigor of its measurements. The team combined structured illumination microscopy with knock-in fluorescently tagged endogenous proteins, ratiometric live-cell pH imaging validated against standard buffers, fluorescence recovery after photobleaching to assess condensate dynamics, quantitative RNA FISH, immunostaining and transcriptome-wide analyses. Mass spectrometry datasets were deposited to the ProteomeXchange Consortium and RNA-seq data to the Gene Expression Omnibus, with source data provided for every figure. By connecting a single charged protein domain to the electrochemical state of a nucleolar subcompartment, and that state to the recovery of an entire organelle, the work delivers a mechanistic model in which stress-recruited SRSF1 alkalizes the dark nucleolar caps, stabilizes the pH gradient of the multiphase nucleolus and enables the cell’s ribosome factory to resume production. It is a vivid demonstration that the chemistry of condensates, not merely their composition, governs how cellular compartments endure and recover from adversity.

Subject of Research: pH regulation of nucleolar condensates by the splicing factor SRSF1 during stress recovery

Article Title: SRSF1 modulates the dark nucleolar cap pH to restore nucleolar integrity and function

Article References: Ma, S., Guo, J., Zhan, X., Wu, F., Yang, S., Huang, C., & Wang, B. (2026). SRSF1 modulates the dark nucleolar cap pH to restore nucleolar integrity and function. Nature Chemical Biology. https://doi.org/10.1038/s41589-026-02316-9

Image Credits: AI Generated

DOI: 10.1038/s41589-026-02316-9

Keywords: nucleolus, SRSF1, pH gradient, biomolecular condensates, dark nucleolar caps, DDX18, RS domain, ribosome biogenesis, cellular stress, phase separation, arginine-rich dipeptides, chemical biology