Human Genes Sing Different Tunes in Different Tissues

Biologists find almost all genes express multiple messenger RNAs Sunday, 02 November 2008 Scientists have long known that it's possible for one gene to produce slightly different forms of the same protein by skipping or including certain sequences from the messenger RNA. Now, an MIT team has shown that this phenomenon, known as alternative splicing, is both far more prevalent and varies more between tissues than was previously believed. Nearly all human genes, about 94 percent, generate more than one form of their protein products, the team reports in the Nov. 2 online edition of Nature. Scientists' previous estimates ranged from a few percent 10 years ago to 50-plus percent more recently. "A decade ago, alternative splicing of a gene was considered unusual, exotic … but it turns out that's not true at all — it's a nearly universal feature of human genes," said Christopher Burge, senior author of the paper and the Whitehead Career Development Associate Professor of Biology and Biological Engineering at MIT. Burge and his colleagues also found that in most cases the mRNA produced depends on the tissue where the gene is expressed. The work paves the way for future studies into the role of alternative proteins in specific tissues, including cancer cells. They also found that different people's brains often differ in their expression of alternative spliced mRNA isoforms. Human genes typically contain several "exons," or DNA sequences that code for amino acids, the building blocks of proteins. A single gene can produce multiple protein sequences, depending on which exons are included in the mRNA transcript, which carries instructions to the cell's protein-building machinery. Two different forms of the same protein, known as isoforms, can have different, even completely opposite functions. For example, one protein may activate cell death pathways while its close relative promotes cell survival. The researchers found that the type of isoform produced is often highly tissue-dependent. Certain protein isoforms that are common in heart tissue, for example, might be very rare in brain tissue, so that the alternative exon functions like a molecular switch. Scientists who study splicing have a general idea of how tissue-specificity may be achieved, but they have much less understanding of why isoforms display such tissue specificity, Burge said. Scientists have also observed that cells express different isoforms during embryonic development and at different stages of cellular differentiation. Burge's team is now studying cells at various stages of differentiation to see when different isoforms are expressed. Isoform switching also occurs in cancer cells. One such switch involves a metabolic enzyme and contributes to cancer cells burning large amounts of glucose and growing more rapidly. Learning more about such switches could lead to potential cancer therapies, Burge said. Until now, it has been difficult to study isoforms on a genome-wide scale because of the high cost of sequencing and technical issues in discriminating similar mRNA isoforms using microarrays. The team took mRNA samples from 10 types of tissue and five cell lines from a total of 20 individuals, and generated more than 13 billion base pairs of sequence, the equivalent of more than four entire human genomes. The sequencing was done by researchers at biotech firm Illumina, using a new high-throughput sequencing machine. Reference: Alternative isoform regulation in human tissue transcriptomes Eric T. Wang, Rickard Sandberg, Shujun Luo, Irina Khrebtukova, Lu Zhang, Christine Mayr, Stephen F. Kingsmore, Gary P. Schroth & Christopher B. Burge Nature advance online publication 2 November 2008, doi:10.1038/nature07509 See also: Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing Qun Pan, Ofer Shai, Leo J Lee, Brendan J Frey & Benjamin J Blencowe Nature Genetics, Published online: 2 November 2008, doi:10.1038/ng.259 ......... ZenMaster

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Panoramic View of Protein-RNA Interactions in Living Cells

Panoramic View of Protein-RNA Interactions in Living Cells Sunday, 02 November 2008 DNA, it has turned out, is not all it was cracked up to be. In recent years we learned that the molecule of life, the discovery of the 20th century, did not – could not – by itself explain the huge differences in complexity between a human and a worm. Forced to look elsewhere, scientists turned to RNA, a direct yet more complex transcript of DNA. But methodological problems have historically plagued the study of RNA regulation in living cells, limiting not only the accuracy of results but also our understanding of RNA's role in human disease. But now, in research to appear in the November 2 advance online issue of Nature, Robert B. Darnell, head of the Laboratory of Molecular Neuro-oncology at Rockefeller University and a Howard Hughes Medical Institute investigator, and his team have changed all that. By adapting techniques mastered in the test tube and combining them with high throughput technology, the team has developed a genome-wide platform to study how specialized proteins regulate RNA in living, intact cells. The platform allows researchers to identify, in a single experiment, every sequence within every strand of RNA to which proteins bind. The result is an unbiased and unprecedented look at how differences in RNA can explain how a worm and a human can each have 25,000 genes yet be so different. "RNA offers a way to make the cell much more complex than what this limited set of genes can offer," says Darnell, who is Robert and Harriet Heilbrunn Professor at Rockefeller. "But how is RNA being regulated in different conditions and diseases, and in different cell types? With this platform, we now have a way to address all these questions." Traditional methods used molecules to extract protein-RNA complexes from living tissue. But often the molecule only extracted the RNA. Other times, the protein bound too weakly to survive the purification process, which involved stripping the complex of unwanted debris. To address the issue, Darnell and his team used a trick from test-tube biochemistry that molecularly cements these regulatory proteins to RNA at the moment they touch. The technique, when applied to high throughput sequencing, is called high throughput sequencing-cross linking immunoprecipitation, or HITS-CLIP for short. Since the RNA and RNA-binding protein are fused together, the researchers can really beat up the extract and rigorously purify the protein without fear of losing the RNA. At the end of the day, they are left with the RNA sequence to which the protein was bound. They can then take these sequences to Rockefeller's high throughput sequence facility, and with the help of Research Support Specialist Scott Dewell, overlay them onto the genome and see where they match. What they get is a map of every position on every transcribed RNA where the RNA binding protein is binding. When DNA is transcribed into RNA, the primary transcript is divided into many blocks called exons, which are separated by empty spaces. In order to convert the transcript into some sort of message, all the spaces need to be removed; but if an exon is dropped, a different version of that protein, which could carry a very different message, is created. "That's RNA splicing," says first author Donny Licatalosi, a postdoctoral associate in the lab. "It is what gives rise to this massive pool of diverse and complex tissues with a relatively small number of genes." In the past, the group used a sophisticated process of evidence and inference to make predictions of the points of regulation along the transcript. "Now, we have direct biochemical validation that these interactions occur in the brain to regulate splicing," says Licatalosi. "The observed map – and this was amazing – looked just like our predicted map," says Darnell. Darnell, Licatalosi and their colleagues Aldo Mele, a research assistant, John Fak, a research assistant, Sung-Wook Chi, a graduate fellow in computational biology and medicine, Xuning Wang, assistant director of biocomputing and Jennifer Darnell, a research associate professor, looked at an RNA-binding protein called Nova2 that is found exclusively in neurons. They found that depending on where Nova2 binds to RNA, they could predict and directly observe whether an exon would be included or excluded in the final transcript, and which protein version it created. "The cell seems to be going through great trouble to regulate these RNAs in different conditions and different cell types," says Darnell. "When RNA developed the ability to make a more stable copy of itself – DNA – it didn't write itself off as a relic for the textbooks. It stayed at the core of complex processes in the cell." Reference: HITS-CLIP yields genome-wide insights into brain alternative RNA processing Donny D. Licatalosi, Aldo Mele, John J. Fak, Jernej Ule, Melis Kayikci, Sung Wook Chi, Tyson A. Clark, Anthony C. Schweitzer, John E. Blume, Xuning Wang, Jennifer C. Darnell & Robert B. Darnell Nature advance online publication 2 November 2008, doi:10.1038/nature07488 ......... ZenMaster

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Mending Broken Hearts with Tissue Engineering

New scaffold approach could also aid engineering of other tissues Sunday, 02 November 2008 Broken hearts could one day be mended using a novel scaffold developed by MIT researchers and colleagues. The idea is that living heart cells or stem cells seeded onto such a scaffold would develop into a patch of cardiac tissue that could be used to treat congenital heart defects, or aid the recovery of tissue damaged by a heart attack. The biodegradable scaffold would be gradually absorbed into the body, leaving behind new tissue. The accordion-like honeycomb scaffold, to be reported in the Nov. 2 online edition of Nature Materials, is the first to be explicitly designed to match the structural and mechanical properties of native heart tissue. As a result, it has several advantages over previous cardiac tissue engineering scaffolds. Further, the MIT team's general approach has applications to other types of engineered tissues. "In the long term we'd like to have a whole library of scaffolds for different tissues in need of repair," said Lisa E. Freed, corresponding author of the paper and a principal research scientist in the Harvard-MIT Division of Health Sciences and Technology (HST). Each scaffold could be tailor-made with specific structural and mechanical properties. "We're already on the way to a few other examples," Freed said. With respect to the current work, "previous scaffolds did not necessarily possess structural or mechanical properties consistent with the native myocardial [heart muscle] structure," said George C. Engelmayr Jr., lead author of the paper and an HST postdoctoral fellow. Heart muscle, he explained, is "directionally dependent" — meaning its cells are aligned in specific directions. The researchers reasoned that "borrowing more closely from nature's lessons," as they write in Nature Materials, might lead to a tissue with properties closer to the real thing. So, using a laser similar to that used for eye surgery, they created a scaffold with directionally dependent structural and mechanical properties. The scaffold has three principal advantages over its predecessors. First, its mechanical properties closely match those of native heart tissue. For example, it is stiffer when stretched circumferentially as compared to longitudinally. Engelmayr found that he could essentially "dial in" specific mechanical properties for the polymer scaffold by varying the time it is allowed to set, or cure. He noted that with this ability, coupled with the flexibility of the laser technique, "we might be able to come up with even better pore shapes with better mechanical properties." In a second advantage, the team found that a patch of tissue created from neonatal rat heart cells cultured on the scaffold showed directionally dependent electrophysiological properties similar to native tissue. In other words, when an electrical field was applied the engineered patch contracted more readily in one direction than in another. In a third advantage, "the scaffold itself has an intrinsic ability to guide the orientation of cultured heart cells," Freed said. Freed was part of another MIT team that, in 2004, showed that heart cells cultured on a traditional scaffold could also be coaxed into alignment, but only with electrical stimulation. The researchers note that the scaffold used in the experiments described above has some limitations. For example, they write, it is "too thin to address reconstruction of full-thickness myocardium." However, as they report in Nature Materials, they have already begun addressing those problems by creating new honeycomb scaffolds that, among other things, allow much thicker, multi-layered tissue structures. Reference: Accordion-like honeycombs for tissue engineering of cardiac anisotropy George C. Engelmayr, Jr, Mingyu Cheng, Christopher J. Bettinger, Jeffrey T. Borenstein, Robert Langer & Lisa E. Freed Nature Materials, Published online: 2 November 2008, doi:10.1038/nmat2316 ......... ZenMaster

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