Posts Tagged ‘protein expression’

irrelevant RNA found to be crucial for protein selection

 :: Posted by American Biotechnologist on 03-21-2011

If a big bunch of your brain cells suddenly went rogue and decided to become fat cells, it could cloud your decision-making capacity a bit. Fortunately, early in an organism’s development, cells make firm and more-or-less permanent decisions about whether they will live their lives as, say, skin cells, brain cells or, well, fat cells.

Those decisions essentially boil down to which proteins, among all the possible candidates encoded in a cell’s genes, the cell will tend to make under ordinary circumstances. But exactly how a cell chooses its default protein selections from an overwhelmingly diverse genetic menu is somewhat mysterious.

A new study from the Stanford University School of Medicine may help solve the mystery. The researchers discovered how a particular variety of the biomolecule RNA that had been thought to be largely irrelevant to cellular processes plays a dynamic regulatory role in protein selection. In unraveling this molecular mechanism, the study also offers enticing clues as to how certain cancers may arise.

Howard Chang, MD, PhD, associate professor of dermatology, is the senior author of the study, published online March 20 in Nature.

“All the cells in your body have the same genes, but they don’t all make the same proteins,” said Chang, MD, PhD, who is also a Howard Hughes Medical Institute Early Career Scientist.

In this new study, Chang and his colleagues identified a novel action by a subset of RNA that reinforces cells’ decisions about which combinations of their genes are to be active and which must stay silent.

RNA is a chemical lookalike of DNA — the stuff our genes are made of — that, according to standard textbooks, mainly functions as a messenger: a copy of a gene, made by a cell’s gene-reading machinery, that can float away from the chromosomes where genes reside to other places in the cell where proteins are made. There the messenger-RNA molecule serves as an instruction manual for the production of proteins.

Scientists used to see RNA mostly as a stodgy servant of its kingly commander, DNA, in the protein-production process. But in recent decades scientists have learned of several ways RNA can influence the production of proteins besides merely conveying information from genes to a cell’s protein-making apparatus.

In the Nature study, the researchers identified a novel regulatory role for a class of RNA molecules called lincRNA (for long intergenic noncoding RNA). A typical cell spawns as many as 10,000 distinct species of lincRNA molecules — on a par with the number of conventional protein-coding genes — but lincRNAs don’t spell out recipes for making proteins. For years, many biochemists were skeptical that lincRNA played any important role in a cell and considered the molecules just mere “noise,” perhaps vestigial protein-coding genes that had mutated to become nonfunctional. Chang’s group has been instrumental in proving that lincRNAs can play a critical regulatory role: determining what proteins a cell produces and, thereby, what identity it assumes.

To do so, Chang and his associates turned to human fibroblasts, which are easily grown in culture. Fibroblasts are cells that lie just beneath the skin and secrete factors determining skin cells’ local character. “You’ll never see hair growing out of someone’s palm,” Chang said. The factors that fibroblasts secrete vary depending on where in the body they’re located.

Remarkably, cultured fibroblasts from different parts of the body somehow remember their sense of where they belong, continuing to maintain characteristic patterns of genes that are “on” or “off” even over dozens of generations of cell division in a petri dish. “Why is that?” Chang asked.

A related question intrigued the study’s first author, Kevin Wang, MD, PhD, an instructor of dermatology and a postdoctoral scholar in Chang’s lab. “I was initially interested in conditions like psoriasis, a skin disease whose manifestations in the body are region-specific,” he said. “Cells that have the same DNA, that look the same under a microscope — what made them act differently?”

Chang has been using cultured fibroblasts as workhorse cells to help answer these questions. In a study published last year in Science, his group showed that one species of lincRNA, which he and his labmates had discovered and named HOTAIR, acts quite differently from your standard mRNA molecule: It contorts into a kind of adapter plug and then latches onto massive protein complexes, which have the ability to silence genes. Once hooked up to such complexes, HOTAIR shuttles them to particular spots along a chromosome — “positional identity” genes. Defects in these genes, first identified in fruit flies, can result in bizarre outcomes such as a fly with legs growing out of its head, instead of antennae. Particular on/off patterns of a cell’s positional-identity genes lead the cell to behave in a characteristic way (palm versus scalp, for example).

In a nutshell, HOTAIR locks cells’ positional identities into place by marking key genes with the biochemical equivalent of “gone fishing” signs, so that they remained closed for business.

The new study, in contrast, demonstrates how another lincRNA, dubbed HOTTIP, grabs onto an opposing type of protein complex, which marks similar positional-identity genes as “open for business.” The researchers observed that this complex wheels into action once HOTTIP links to it, and then biochemically fixes cell-position-appropriate genes in the “on” position.

An ability to act as a mute button for protein production has been demonstrated for other RNA types besides lincRNA. But, said Chang, HOTTIP is the first example of any RNA molecule that creates a memory of gene activation rather than silencing them. “When we experimentally impeded HOTTIP activity, fibroblasts that were supposed to express certain positional-identity genes didn’t,” he said.

Interestingly, the particular genes that HOTTIP caused to retain a switched-on status were fairly remote from one another along the stretch of chromosome where they reside. To learn more about how this works, Chang, Wang and their Stanford colleagues teamed up with a group at the University of Massachusetts Medical School, in Worcester, whose research focuses on the three-dimensional organization of genomes.

What they learned from this holds implications for how some cancers could get started. The investigators found that DNA can form complicated looping structures that bring genes distant from one another on a chromosome, or on entirely different chromosomes, physically close. This lets HOTTIP and the protein complex it’s linked to efficiently mark appropriate genes as “open for business.”

But it could also lead to things going awry, possibly leading to certain cancers. Biochemical interactions at close range among these ordinarily distant genes can cause their fusion — or even an exchange in their positions — and resulting faulty protein production characteristic of a number of cancers, Chang said.

The study was funded by the California Institute for Regenerative Medicine, the National Institutes of Health, the Scleroderma Research Foundation, the W.M. Keck Foundation and the Howard Hughes Medical Institute. Other Stanford co-authors are Joanna Wysocka, PhD, assistant professor of chemical and systems biology and of developmental biology; Jill Helms, PhD, DDS, professor of surgery; Rajnish Gupta, MD, PhD, clinical assistant professor of dermatology; Bo Liu PhD, a research associate in Helms’ laboratory; medical and graduate student Yul Yang; graduate student Ryan Corces-Zimmerman; medical student Ryan Flynn; and research assistant Angeline Protacio. In addition to the team at the University of Massachusetts, the study also involved a researcher at the University of Michigan.

Source: Stanford School of Medicine

Altering Protein Production with Alu Elements

 :: Posted by American Biotechnologist on 02-28-2011

Part of the answer to how and why primates differ from other mammals, and humans differ from other primates, may lie in the repetitive stretches of the genome that were once considered “junk.”

A new study by researchers at the University of Iowa Carver College of Medicine finds that when a particular type of repetitive DNA segment, known as an Alu element, is inserted into existing genes, they can alter the rate at which proteins are produced — a mechanism that could contribute to the evolution of different biological characteristics in different species. The study was published in the Feb. 15 issue of the journal Proceedings of the National Academy of Sciences (PNAS).

“Repetitive elements of the genome can provide a playground for the creation of new evolutionary characteristics,” said senior study author Yi Xing, Ph.D., assistant professor of internal medicine and biomedical engineering, who holds a joint appointment in the UI Carver College of Medicine and the UI College of Engineering. “By understanding how these elements function, we can learn more about genetic mechanisms that might contribute to uniquely human traits.”

Alu elements are a specific class of repetitive DNA that first appeared about 60 to 70 million years ago during primate evolution. They do not exist in genomes of other mammals. Alu elements are the most common form of mobile DNA in the human genome, and are able to transpose, or jump, to different positions in the genome sequence. When they jump into regions of the genome containing existing genes, these elements can become new exons — pieces of messenger RNAs that carry the genetic information.

Although scientists have known for more than a decade that these Alu elements are an important source of new exons in the human genome, it has been more difficult to determine if these new exons are biologically important.

“It’s been hard to say whether these Alu-derived exons actually do anything on a genome-wide level,” Xing said. “Our new study says they do – they affect protein production by altering the efficiency with which messenger RNA is translated into protein.”

Xing noted that in other circumstances, altering the rate of protein production can cause disease, meaning that a mechanism that can affect protein production can have a real impact on the characteristics of an organism.

“This would not be the only mechanism that might differentiate humans from other primates, but our study suggests that the creation of new exons from Alu elements is an important process that contributes to those differences,” Xing said.

The UI team, including co-first authors Shihao Shen, doctoral student in the Department of Biostatistics; and Lan Lin, Ph.D., associate in the Department of Internal Medicine, made use of data from a new technology called high throughput RNA sequencing to analyze more than 120 million RNA sequences from human cerebellum. Using this data, the team was able to quantify how often Alu-derived exons were included in the mature RNA sequences, which provide the final blueprint for protein production, and where they were inserted in the genes.

“What we found is that these exons tend to avoid protein-coding regions of the genes and rather they end up in the non-coding region that precedes the protein-coding region, called the five prime untranslated region or 5′ UTR,” Xing explained. “This is the part of the gene that usually contains regions that help control the stability of the messenger RNA and the efficiency at which the messenger RNA is translated into protein.”

Experiments to probe the function of these newly inserted elements proved that Alu exons in this region are able to alter the efficiency of messenger RNA translation, which means they affect how fast protein is produced from the altered genes.

The study also suggests that the effect of the newly created exons might be amplified because of which genes were “targeted” by the Alu exons. The researchers found that Alu exons are highly enriched in genes that code for zinc-finger transcription factors — proteins that act as master regulators of gene expression and that previously have been linked to human and primate evolution. Because these transcription factors control the expression of thousands of other genes, any changes to the amount of transcription factor available would likely have a cascade effect on the downstream genes.

Source: Press Release

Sustainable RNA Transfection

 :: Posted by American Biotechnologist on 07-26-2010

MIT scientists Matthew Angel and Mehmet Fatih Yanik have discovered a method for transfecting mRNA into fibroblasts without triggering the immune response that normally defends cells against exogenous RNA infection. Cells are usually able to differentiate between endogenous and exogenous RNA through activation of pattern-recognition receptors (PRRs) that initiate a subsequent immune response. While this immune response is important for defending cells against unwanted viral RNA invasion, it also serves as a barrier for scientists interested in delivering protein-encoding mRNA into cells for a variety of purposes.

Why the need to deliver mRNA into cells? Why not just deliver DNA as is normally done utilizing traditional transfection techniques? Or, better yet, why not just skip the translational step altogether and deliver protein directly to the cells?

According to the study’s authors, since DNA is incorporated into the genome it runs the risk of causing genomic disruption which may limit its therapeutic potential while protein transduction tends to be an expensive and inefficient process. In their extensive review on the use of RNA vaccines in cancer treatment, Bringmann et al discuss how RNA has several advantages including its ability to be generated in large quantities, to be easily degraded, to not be integrated into the genome and to be quickly cleared out of the organism. As such, RNA has the potential to be a great therapeutic tool that is unencumbered by some of the pathogenic risks inherent in other molecular therapies.

Yet, obtaining sustained protein expression following mRNA transfection is challenged by the innate immune response generated by exogenous RNA. In this study, published in PLoS ONE earlier this month, Angel and Yanik show how an siRNA cocktail of interferon-β (Ifnb1), Eif2ak2, and Stat2 inhibits the RNA-mediated immune response and enables repeated mRNA transfection and subsequent protein expression.

While this particular study focused on reprogramming techniques to generate autologous pluripotent stem cells, the methodology could likely be utilized in a variety of experimental conditions focused on obtaining expression of active proteins in an inexpensive and sustainable manner.

For more information see Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins

Angel M, & Yanik MF (2010). Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins PLoS ONE, 5 (7)