PerspectiveMedicine

The Cart Before the Horse

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Science  05 Sep 2008:
Vol. 321, Issue 5894, pp. 1302-1304
DOI: 10.1126/science.1163791

Since the identification of specific regions in human chromosomes that undergo recurring structural rearrangements (translocations) and cloning of the associated breakpoint genes, the fusion of genes has been viewed as a unique event in abnormally growing, usually malignant, cells (1, 2). However, the study by Li et al. on page 1357 in this issue (3) indicates quite the contrary, turning at least one paradigm of cancer cytogenetics on its head.

Li et al. report that in normal human endometrial tissue, there is a low amount of a messenger RNA (mRNA) that corresponds to sequences from two genes, JAZF1 on chromosome band 7p15 and JJAZ1/SUZ12 on chromosome band 17q21. Moreover, this chimeric mRNA is identical to that seen in 50% of human endometrial stromal sarcomas, in which there is a 7;17 chromosomal translocation that results in a gene fusion, even though no translocation is detected in normal endometrial cells. The product encoded by the chimeric mRNA is a fusion protein that is expressed in cultured cells, and consequently, could confer cellular resistance to programmed cell death and increased growth (under conditions where expression of the endogenous JJAZ1 gene was suppressed). Further, this fusion mRNA is expressed in a cyclical manner in normal endometrial cells, most readily detected at the beginning and end of the menstrual cycle when concentrations of estrogen and progesterone are low.

How is this fusion mRNA made in the absence of a corresponding gene fusion? Li et al. propose that the fusion mRNA is produced by trans-splicing of RNA (see the figure) in which nucleotides at the 3′ end of JAZF1-encoding precursor mRNA are replaced with those of JJAZ1-encoding precursor mRNA. A different form of trans-splicing is common in several lower animal phyla (4), and a few mRNAs have been shown to be assembled from separate transcripts in insects (5). And although trans-splicing in mammalian cells has been reported, the resulting chimeric RNAs do not perform obvious functions and are usually not present in large enough amounts to do so.

Fusion RNAs.

Either chromosome translocation or RNA trans-splicing can give rise to fusion mRNAs and proteins. Some chromosomal translocations produce two hybrid genes that may produce mRNAs containing the 5′ end of one gene and the 3′ end of the other. Both may encode fusion proteins. Alternatively, normal mRNAs corresponding to both genes can recombine by trans-splicing that may produce equivalent fusion mRNAs and proteins. Only one of the possible fusion mRNAs and proteins is examined by Li et al.

CREDIT: ADAPTED BY P. HUEY/SCIENCE

It has not been clear how two separate mRNAs are spliced together (6). Perhaps they are brought together through the pairing of nucleotides within noncoding sequences (introns) between two transcripts, or maybe each contains a binding site for proteins that can form dimers or higher-order multimers. Although sloppiness by the spliceosome—the cellular machine that removes introns from precursor RNA—could be an explanation, only specific pairs of precursor mRNAs engage in trans-splicing. Perhaps RNAs from different genes are trans-spliced because they are transcribed in the same geographic location. Alternatively, trans-splicing could occur more frequently than we realize, but most cases go undetected.

What makes the study by Li et al. especially interesting is that trans-splicing is clearly regulated. The mRNA fusion appears only in cells from endometrial tissue. Its expression is increased by hormones and hypoxia, with much higher expression in late secretory and early proliferative stages of the menstrual cycle. The key question is whether the chimeric RNA is transcribed from some undetected rearranged copies of the two genes. However, Li et al. show that there is no such gene rearrangement in cells producing the trans-spliced mRNA and that this trans-splicing event can be duplicated in vitro. In addition, they show that a nontransformed human endometrial stromal cell line had no rearranged DNA or visually abnormal chromosomes.

Given the absence of any detectable rearranged DNA in cells producing the chimeric RNA, the obvious explanation is rearrangement at the RNA level. To demonstrate that trans-splicing could account for the chimera, Li et al. made extracts from a human endometrial stromal cell line and from a rhesus monkey fibroblast cell line so that they could detect trans-spliced products by a sequence difference between the RNA from the two species. The authors demonstrated in vitro trans-splicing of the rhesus JAZF1 exons (coding regions of DNA) to human JJAZ1 exons. Treatment of the rhesus RNA with deoxyribonuclease (to cleave any DNA that might be present) did not prevent formation of the chimeric RNA, confirming that chimeric RNA arose from trans-splicing.

Is it a coincidence that the same RNA occurs in normal cells by trans-splicing and in tumor cells of the same type by DNA rearrangement? The authors suggest the intriguing possibility that whatever leads to the trans-splicing could also lead to the genomic rearrangement. This could occur by at least three general mechanisms. The same sequences could pair at the RNA level to result in trans-splicing and at the DNA level to result in genomic rearrangement. However, sequence analyses of translocation breakpoints in leukemia reveal large deletions and duplications as well as precise nucleotide base-pairing (7). Alternatively, genomic rearrangement could be a direct result of the trans-splicing event if genes involved in the rearrangement are brought into close proximity during the RNA trans-splicing process. This idea is consistent with recent reports on the existence of “factories” for transcription and RNA processing (810). Finally, the RNA created by trans-splicing could act as a guide RNA to facilitate the genomic rearrangement, an idea for which there is precedent (11). In this case, the trans-spliced RNA would anneal to regions of both of the chromosomes and guide them in a DNA recombination event. Indeed, it is possible that other genomic rearrangements could be guided by cellular RNAs.

If fusion mRNA is widespread, it could explain the conundrum that has long perplexed cancer geneticists: why fusion mRNAs can be detected in apparently normal tissues of healthy people. Such fusions involve common translocations seen in neoplastic hematopoietic cells, but never in solid tumors. If fusion mRNAs are part of normal cell function, then finding fusions of the immunoglobulin heavy chain gene (IGH) to the BCL2 gene in normal spleens, which usually reflects the presence of a t(14;18) translocation in lymphomas, would not be unexpected (12). Given that IGH and the MYC genes frequently colocalize in transcription factories (9), this geography could provide a mechanism for having nascent RNAs in juxtaposition; moreover, the genes themselves would be close together. Translocations involving the IGH and IGK/L genes and the genes encoding T cell receptors (TCRs) in lymphoid malignancies are exceptions in that they do not lead to a fusion mRNA, but rather to altered regulation of the apparently normal target protein (13). Presumably all translocations are mediated by DNA recombination enzymes, but could this process be guided by RNA produced by trans-splicing? The study by Li et al. also raises questions relevant to clinical practice. Potent therapies targeting fusion mRNA and proteins may disrupt critical pathways of normal cell function. Increasingly sensitive methods to determine the presence of a few translocation-bearing cells leads one to question whether translocations or normal cell products are being detected. This is a critical issue because the search for minimal residual disease is in high gear, especially for chronic myeloid leukemia that responds to the drug imatinib, as a “cure” seems within reach (14). Many patients suffering from this cancer are translocation-negative on standard cytogenetic analysis, but show a gene fusion (BCR-ABL) by reverse transcriptase polymerase chain reaction. For these patients, especially the ones with very low amounts of fusion, it is unclear whether what is being detected is a malignant cell or a trans-splicing event.

As the search for fusions in normal cells will likely be fast-paced for the next few years, two points should be considered, given the findings of Li et al: cell specificity and regulation of the trans-splicing event. So choose the fusion to be investigated, mindful of these constraints.

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