Preprint: enlarged cells have less MDM4, a regulator of p53, which halts cell division

Preprint: enlarged cells have less MDM4, a regulator of p53, which halts cell division

A preprint published on bioRxiv on October 22 examines how excessive cell growth helps sustain cell division arrest. The authors used the human RPE1 cell line and separately tested the role of MDM4, a protein that normally restrains p53.

A senescent cell stops dividing for an extended period and often grows larger. The authors tested whether cell size itself helps maintain this state.

RPE1 cells were held for seven days in G1, the phase before DNA replication. Some cells continued to grow during this time. In two control conditions, cells were kept close to their usual size by limiting growth with Torin1 or shortening the arrest to two days. This comparison separates the effects of cell size from those of arrest duration and treatment with a single drug. Enlarged cells had higher nuclear levels of p53 and p21: p21 relays this signal and prevents cells from proceeding to DNA replication. Of the 342 p53 RNA targets measured, 142 increased significantly.

Nuclear organization also changed. Nuclear speckles, regions where messenger RNAs mature, became larger and more sparsely distributed throughout the nucleus. Chromatin proximity maps showed that genes normally located closer to speckles moved farther away and became less active. Some genes near the nuclear lamina, a layer along the edge of the nucleus, moved closer to speckles and became more active.

Enlarged cells also showed changes in splicing, the process of assembling mature messenger RNA from segments. In MDM4 RNA, the sixth exon was skipped more often: the proportion of the shortened variant MDM4-S increased more than ninefold. This RNA is rapidly degraded, reducing the amount of full-length MDM4. When MDM4 was reduced in a separate experiment, p53 and p21 increased, and fewer cells entered S phase, when DNA replication begins.

Restoring full-length MDM4 in enlarged cells reduced p21, helped cells exit G1, and improved their growth in a long-term experiment. Delays at later stages of the cell cycle associated with replication stress persisted. In this cell model, the authors functionally tested the role of MDM4; they observed nuclear reorganization and changes in splicing in the same enlarged cells.