Recurrent chromosomal translocations are a genetic hallmark for many types of human cancer. They are mostly generated by NHEJ-mediated pathways of DNA double-strand breaks that occur in selected genes of our genome and which seem to be prone to DNA damage (genetic hot spots). Here, we demonstrate that genes known to be involved in chromosomal translocations display a genuine and novel feature: early-termination of transcriptional processes. Early termination was observed in those regions of the investigated genes that have already been defined as breakpoint cluster regions. The abundance of these early terminated transcripts was in the range of 10-20% when compared to their corresponding full-length transcripts. These shorter transcripts seem to participate in intragenic and intergenic trans-splicing events, resulting in the creation of RNA species that exhibit exon-repetitions or resemble chimeric fusion RNAs. All our data were obtained by using human peripheral blood mononuclear cells (PBMCs) deriving from healthy individuals. These data confirm earlier studies made by others [15
], however, we present for the first time a rational explanation for this unusual observation.
In first instance, the production of early-terminated transcripts seems to provide negative effects to cells, because it uses predominantly transcripts of the same gene to saturate the unique splice donor site, and thus, is lowering the transcript abundance of such genes. Moreover it also disrupts transcripts of other genes due to trans-splicing events. On the other hand, trans-splicing of RNA molecules remind us of the exon-shuffling mechanism that turned out to be a useful mechanism to create novel genes from existing ones during evolution [41
However, the question remains what is causing early-termination of transcription. Cryptic poly-A sites, transcriptional pausing/stalling due to specific chromatin features or the presence of repetitive DNA elements may provide possible explanations. Another explanation is provided by microRNAs. A recently published study identified microRNAs encoded at a genomically unstable region [42
]. Such microRNAs can specifically bind to their own gene transcripts and probably cause cleavage. The outlined mechanisms or any combination thereof possibly explain the creation of early-terminated transcripts, exhibiting a non-saturated splice donor site, and therefore, produce molecule species that are able to initiate in
cis splicing processes to cryptic exons (if available) or participates in trans-splicing reactions.
This study demonstrated that early-terminated transcripts seem to be the molecular source for the creation of trans-spliced RNA species. Most trans-spliced RNA species result in exon-repetitions (intragenic trans-splicing; identified in RT-PCR experiments) while only few cause the formation of chimeric fusion transcripts (intergenic trans-splicing; identified in nested RT -PCR experiments). In addition, most identified trans-spliced fusion RNAs displayed no continuous open reading frame (ENL-RPL18A, ENL-CCDC127, ENL-SFPQ, ELL-MKI67, ELL-SFRS7, and ELL-RPS6). By contrast, we identified ENL-ALKB7, MLL-AF4 and NPM-ALK with fused open reading frames in PBMCs of healthy individuals. Noteworthy, the latter two are identical to chimeric fusion transcripts that are regularly identified in tumor cells carrying the specific chromosomal translocations t(4;11)(q21;q23) and t (2;5)(p23;q35), These translocations are all associated with acute lymphoblastic leukemia and anaplastic large cell lymphoma (ALCL), respectively. Therefore, we suggest defining these chimeric fusion RNAs produced in non-rearranged cells as “pro-neoplastic RNA molecules".
The existence of pro-neoplastic RNA molecules in healthy individuals is not restricted to hematopoietic cells. Recent findings suggest that chimeric fusion RNAs can be produced in healthy (by trans-splicing) and in cancer cells carrying indeed this particular genetic rearrangements. Examples are the JAZF1-JJAZ1
chimeric fusion RNA in normal endometrial cells [43
], or the androgen-induced SLC45A3-ELK4
chimeric fusion RNA in normal prostate cells [44
]. The JAZF1-JJAZ1 fusion protein provides anti-apoptotic activity, indicating that such a trans-spliced fusion RNA even provide a benefit to those cells. Moreover, the chimeric fusion RNAs seem to be produced in a cell-type specific manner [43
]. This could be explained by a cell-type specific localization of chromosome territories in the nucleus; because chromatin loops deriving from different chromosomes must be attracted to a common transcription factory [33
] in order to create such trans-spliced fusion RNAs.
These findings are leading back to our initial question whether there exists a molecular mechanism that could explain how illegitimate recombination events are created and why the phenomenon of recurrence exists in human tumor samples. Are pro-neoplastic RNA molecules somehow involved in the creation of illegitimate recombination? If so, the trans-spliced fusion RNA molecules must be able to influence DNA repair processes. This idea is supported by three recent findings:
- Storici and coworkers demonstrated for the first time that DNA repair in fission yeast could be templated by small RNA molecules . The existence of “RNA-templated DNA repair” in fission yeast implies that RNA has per se the capability to influence DNA repair processes, most likely by basepairing to DNA sequences flanking a damaged DNA region.
- Another study revealed that a component of the NHEJ machinery, DNA ligase IV, has the ability to catalyze a ligation reaction between single-stranded DNA ends . Thus, the opposite DNA strand - not basepairing to a complementary, chimeric fusion RNA - could be ligated by using the single-stranded DNA ends of a broken DNA molecule.
- Thirdly, the ENCODE project  has already demonstrated that hnRNA molecules seem to be generated from most parts of the human genome, including long intergenic regions. Unspliced hnRNA molecules represent perfect copies of genomic regions and are easily accessible for DNA repair processes, without the need to find appropriate DNA sequences on a homologous chromosome.
Based on these data and our findings, we propose a hypothesis that might explain the onset of chromosomal translocations: (a) transcription of genes in local proximity, e.g. in a common transcription factory, (b) the presence of early-terminated transcripts in certain genes of our genome, (c) the creation of trans-spliced fusion transcripts, (d) the occurrence of DNA double-strand breaks in both genes involved in genetic recombination events, and finally (e) the formation of RNA/DNA hybrid structures that guide subsequent DNA repair process. The latter process is depicted in . It is important to note that the trans-spliced chimeric RNA molecules, in this case the MLL-AF4
fusion RNA, is not a substrate for DNA repair polymerases rather than forcing an “RNA/DNA repair structure". Due to the enzymatic activity of DNA ligase IV, the genomic fusion may occur at any sequence downstream of MLL
exon 9 (e.g. MLL
intron 9) and upstream of AF4
exon 4 (e.g. AF4
intron 3). This is exactly where most t(4;11) leukemia patients display their chromosomal fusion site. Thus, the most abundant trans-spliced MLL (exon9): AF4(exon4)
fusion RNA is explaining also the breakpoint distribution that has been described for acute leukemia patients bearing t(4;11) translocations [52
Figure 4 Proposed hypothesis for the RNA-guided DNA repair mechanism. A. Double-stranded DNA breaks downstream of MLL exon 9 and upstream of AF4 exon 4 (or exon 5) are a genetic pre-requiste for misguided DNA repair and the onset of CTLs. B. The transspliced RNA, (more ...)
The existence of an “RNA-mediated proof-editing process” would not only be interesting for the onset of specific genetic lesions, but could be a novel and fundamental mechanism for maintaining genome integrity by using the existent hnRNA molecules. We are currently conducting experiments that aim to validate this novel hypothesis and to investigate these emerging data in more detail.