In the past there have been only few experimental approaches linking Alu DNA elements and their sequences, such as the internal poly A tail, to disease.2,17,18
In the present study we focused on the determination of Alu transcripts which have been functionally discussed as modifiers of translation and, more recently, transcription.4,8
In a reverse experimental approach Bennet et al.9
reported on a data base search for putative active, i.e., transcribed, Alu DNA elements. The authors subsequently confirmed activity by retrotransposition via RNA in HeLa cell culture. At the same time, we had already identified in a “real-time experimental approach” Alu transcripts and assigned them to human chromosomes. To produce reliable results we established robust protocols for Alu transcript identification and characterization. This approach revealed different Alu-specific transcriptional activity in leucocytes from humans including sCJD, dementia and AD cases. Our aim was to investigate small non-coding RNA since such transcripts were hypothesized to play a role in prion disease.19
In Alu DNA element research transcripts of the Alu elements have never before been analyzed in a real-time experimental approach. Our findings are thus promising and novel for Alu transcription in human blood cells.
Working with Alu transcripts is drastically hampered by any trace amounts of genomic DNA that may contaminate RNA preparations. Since small-sized RNA may be lost by silica-based column methods we applied phenol-type precipitation methods to isolate RNA. Tests for DNA contamination were performed with appropriate β-actin primer pairs as single copy gene control, and RNA was separated on agarose gels to visualize the integrity of RNA preparations treated with DNase. We used the dUTP/ UNG method to prevent carry-over; this, however, required plasmid replication/propagation in UNG negative cells that were not prone to degrade cloned Alu cDNA sequences containing U instead of T. After developing these controls, we ventured an interpretation of our sequence data derived from RNA, i.e., the cloned cDNA.
Our data clearly pointed to a dominance of transcripts from “younger” Alu Y elements over those belonging to the S-family.20
Yet transcripts from older Alu family families were also identified, similar to previous reports on Alu transcripts from “old” Alu elements after virus infection.21
The presence of nucleotide G at position 24/25 of the reference Alu DNA element sequence confirmed our interpretation that we were dealing with Alu transcripts from transcriptionally active Alu DNA elements. In addition to this G we detected a putative “GCACUU” target sequence for a micro RNA around position 34 (unpublished, Kiesel et al.).
An obvious drawbrack in this first study is the lack of statistics. Although we had enough cases and controls to generate Alu RNA/cDNA clones, their number did not allow to statistical confidence. We therefore do not yet suggest active Alu DNA elements, i.e., Alu DNA element transcription patterns, as a marker for neurodegenerative diseases. Larger numbers are necessary for statistical evaluation. Single observations might nevertheless be important for developing of future experimental approaches.
Cloning of Alu transcript amplicons products and “resolving” the amplicon population into single clones revealed a preferred transcription of some Alu DNA elements. Furthermore, the disproportionate transcription of Alu DNA elements present in the human genome could easily be seen. Similar findings were reported for a permanently growing cell line but no one has described this for human Alu transcripts so far.22
It is important to note that some RNA/cDNA clones were observed to align perfectly to several Alu DNA elements on several chromosomes, and are thus most likely Alu DNA element paralogues. In fact, these paralogues may have resulted from recent transposition events. This finding and the fact that preferably Alu Y transcripts have been detected may be indicative for recent transposition of these distinct Alu DNA elements. There was most likely not enough time in evolution to introduce mutations by deamination to counteract retrotransposition. Thus, our observations imply that paralogues of active Alu DNA elements exist and were detectable in our real-time approach. Our results demonstrate how real-time Alu DNA element transcription and retrotransposition occur.
The chromosomal patterns of active Alu DNA elements were divergent when we compared healthy humans with those presenting with neurodegenerative diseases such as sCJD, Alzheimer disease or dementia. We were also aware that we were observing Alu transcription in the periphery but not in the brain where these diseases exert their pathogenic potential.
Unexpectedly, control and sCJD as well as dementia and AD source RNA preparations resulted in Alu RNA/cDNA that could be located to their respective Alu DNA elements in different chromosomes and genomic locations. Indeed, we were able to clearly document that different Alu DNA elements are transcribed in living organisms. Our data were compared with previous results from experiments with permanently growing cells under experimental conditions such as viral infection. 23,24
Divergent transcription patterns may point to different Alu-related transcriptomes in individuals. It may be that additional epigenetic mechanisms are responsible for allowing either RNA polymerase III Alu DNA element transcription or RNA polymerase II transcription of certain genes with embedded Alu DNA element sequences.
Stress may also result in a modified transcriptional program even in peripheral leucocytes from patients with neurodegenerative disease. Thus, Alu transcription profiles may serve as an indicator for differential gene expression similar to what is performed on microarrays with high resolution of distinct sets of genes. Those Alu sequences accessible to transcription may then emerge as a surrogate of the cellular transcriptome. In chromosomes in which more than three Alu RNA/cDNA clones could be positioned our impression was that a genome-wide differential Alu DNA element transcription activity is initiated, e.g., on chromosomes 1, 2 and 3 from controls versus 2 and 17 in sCJD cases, or 2 and 11 in AD cases.
In conclusion, our results represent the first real-time study on Alu transcripts transcribed from “active” Alu DNA elements in peripheral blood cells in healthy humans and humans suffering from neurodegenerative diseases. Future research is necessary to confirm the role of Alu trancripts as modifier of gene expression. Although the results of this study may not serve as a basis for developing a marker for prion diseases, they do broaden our limited knowledge of Alu DNA element transcription in human blood cells.