Light Activated Chloroplast Factor Removes Protein Introns

    A new way of fixing inactive proteins has been discovered in an algae, which uses chloroplast extracts and light to release an interrupting sequence from a protein.

    In a protein, the “weakest link” can be an extra sequence that disrupts the protein’s function. Such sequences, according to research from the Boyce Thompson Institute (BTI), can be removed by a system newly discovered in the algae Chlamydomonas reinhardtii. BTI researchers have shown that chloroplast extracts from C. reinhardtii can accomplish the light-activated removal of interrupting sequences from at least one protein. The researchers anticipate that factors from the extracts could be modified for general use in biotechnology.

    BCI scientists Stephen Campbell and David Stern discovered this new repair system while purifying a protein from the chloroplasts of C. reinhardtii that can cut RNA. Upon sequencing the protein, he identified it as RB47, a protein that was not known to have any RNA-cleaving ability. Dr. Campbell noticed that the middle of the protein was missing. When he compared the protein sequence to its corresponding gene sequence, the protein was much shorter than expected.

    Upon further study, Dr. Campbell found that he could detect a long version of the protein that contained an insertion and a short version that didn’t. The cells make both versions when grown in the light or the dark, but only the short version can cleave RNA. The long version of the protein could be converted into the short one by mixing it in a test tube with chloroplasts from cells grown in the light and by illuminating the reaction. This process removed the interrupting insertion and restored the RNA-cutting activity of the protein. It is likely that the chloroplast maintains the machinery necessary to remove the sequence so that it can restore functionality to the protein.

    Details of the work appeared in the July 29 issue of the Journal of Biological Chemistry, in an article entitled, “Activation of an Endoribonuclease by Non-intein Protein Splicing.” The article described how epitope tags and protein sequencing were used to demonstrate that a nonconserved sequence in the was excised from a full-length version of the RB47 protein, and that the flanking sequences were spliced together.

    “The requirement for endogenous factors and light differentiates this protein splicing from autocatalytic inteins, and may allow the chloroplast to regulate the activation of RB47 endoribonuclease activity,” wrote the article’s authors. “We speculate that this protein splicing activity arose to post-translationally repair proteins that had been inactivated by deleterious insertions or extensions.”

    Because the insertion can be placed so that it interrupts a protein’s function, the insertion and repair system may be useful for producing certain pharmaceuticals or protein products—such as cancer drugs—in culture that would otherwise kill the cell. After purification, the inactive products could be treated with chloroplast factors and light to remove the insertion and activate the proteins.

    In future work, the researchers plan to investigate exactly how the insertion becomes spliced out of the protein and which plant factors facilitate its removal. They also aim to understand the purpose of the insertion, and whether the algae can control the splicing to respond to changes in the environment.

    Campbell and Stern also want to know how widespread this new type of protein splicing might be.

    “If it is happening in plants, is it happening in animals?” said Stern. “We’re pretty sure that this protein is just one example; that we have only found the tip of the iceberg.”