Chronic hypoxia upregulates RNA polymerase I, altering methylation of ribosomal RNA

Hypoxia – where a substance is extracted from an adequate supply of oxygen – is a feature within solid cancer tumors that makes them highly aggressive and resistant to treatment.

Studying how cells respond to this critical stress, by altering their metabolism to survive in a low-oxygen environment, is important to better understand tumor growth and proliferation.

Researchers led by Rajeev Samant, Ph.D., professor of pathology at the University of Alabama at Birmingham, now report that chronic hypoxia, surprisingly, increases RNA activity polymerase I and altering the methylation patterns on ribosomal RNA.

These altered epigenetic markers on the ribosomal RNAs appear to form a pool of specific ribosomes that can regulate messenger RNA translation differently.

This supports a postulate, long debated in biology, that the ribosomal protein factories can be reprogrammed in response to pressures for the manufacture of specific proteins.

Ribosomes are macromolecular mechanisms produced by ribosomal RNA and ribosomal proteins, of which up to 10 million may be contained in mammalian cells.

They read a messenger RNA that directs the ribosome to add specific amino acids, from a selection of 20 different amino acids, to a growing peptide chain that folds to release the finished protein. creation. Messenger RNA is a copy of the DNA gene for that protein, and carries the gene information to build a protein from the chromosome to the ribosomes.

RNA polymerase I activity was not expected to be significantly increased in hypoxic breast tumors, mammary epithelial, and breast epithelial cell lines used as models in UAB study because RNA polymerase I is the only RNA polymerase that was used to produce ribosomal RNA, and was tightly regulated.

Hypoxic cells could be expected to reduce their ribosomal RNA synthesis to save energy, without producing more. But according to more RNA polymerase I found by the UAB researchers in hypoxic cells, there was increased ribosome biogenesis in both hypoxic model cell lines and hypoxic regions of spontaneous tumors in mice, as measured by increased nucleoli and another test.

But were these new ribosomes different from ribosomes found in cells grown below normal oxygen levels?

One major challenge in defining specialized ribosomes is the lack of an approach to the subdivision of ribosomes that can separate multiplicity of action.

To selectively separate the subpopulation, the UAB RNA team used a messenger for a protein called VEGF-C, a protein known to increase the formation of new blood vessels called angiogenesis.

Hypoxia is known to induce conversion to angiogenesis and elevated expression of VEGF-C. Furthermore, during hypoxia, VEGF-C messenger RNA is interspersed with alternate ribosomal entry space, controlled by the internal ribosome entry site, or IRES, sequence on VEGF-C messenger RNA.

Samant and colleagues found that VEGF-C production was induced by hypoxia in their cellular models. They were able to use the VEGF-C IRES to pull down hypoxic ribosomes, and found that these ribosomes had a methylation pattern on their 18S, 28S, and 5.8S ribosomal RNAs that were different from Ribosomal RNAs of cells grown in normal oxygen.

In further detailed work, UAB researchers attempted to discover basic calls that control changes in ribosomal RNA methylation patterns under hypoxia.

Samant and colleagues found that the expression of a protein called NMI significantly compromised protein and messenger RNA levels during hypoxia. NMI contains a previously unidentified RNA recognition motif that can bind single-stranded RNAs.

One class of single-stranded noncoding RNAs in cell nuclei, known as SNORDs, is known to direct the fibrillarin methyltransferase enzyme to methylate specific bases of ribosomal RNA. When UAB researchers examined which RNA strains may be linked to NMI, “to our delight,” Samant said, “we found that many of the RNAs were SNORDs. the high-quality RNA groups associated with NMI. “

There are more than 106 SNORDs in human cells to direct methyltransferase to specific sites; NMI linked nine SNORDs associated with changes at 14 methylation sites on the ribosomal hypoxic RNAs. NMI also bound the fibrillarin methyltransferase.

We hypothesize that NMI works to regulate the pool of available fibrillarin and SNORDs that can interact, influencing rRNA methylation patterns. “

Rajeev Samant, PhD, Professor of Pathology, University of Alabama at Birmingham