Comprehensive analysis of molecular pathology requires a collection of reference samples representing normal tissues from healthy donors. For the available limited collections of normal tissues from postmortal donors, there is a problem of data incompatibility, as different datasets generated using different experimental platforms often cannot be merged in a single panel. Here, we constructed and deposited the gene expression database of normal human tissues based on uniformly screened original sequencing data. In total, 142 solid tissue samples representing 20 organs were taken from post-mortal human healthy donors of different age killed in road accidents no later than 36 hours after death. Blood samples were taken from 17 healthy volunteers. We then compared them with the 758 transcriptomic profiles taken from the other databases. We found that overall 463 biosamples showed tissue-specific rather than platform- or database-specific clustering and could be aggregated in a single database termed Oncobox Atlas of Normal Tissue Expression (ANTE) . Our data will be useful to all those working with the analysis of human gene expression.

Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5 untranslated region (5UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5UTR-Fluc) or bicistronic (Rluc-L1 5UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5UTR. Nevertheless, this cap-dependent initiation activity of the L1 5UTR was unexpectedly high and resembles that of the beta-actin 5UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5UTRs and call into question the conception that every long GC-rich 5UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event.

Many mammalian mRNAs possess long 5′ UTRs with numerous stem-loop structures. For some of them, the presence of Internal Ribosome Entry Sites (IRESes) was suggested to explain their significant activity, especially when cap-dependent translation is compromised. To test this hypothesis, we have compared the translation initiation efficiencies of some cellular 5′ UTRs reported to have IRES-activity with those lacking IRES-elements in RNA-transfected cells and cell-free systems. Unlike viral IRESes, the tested 5′ UTRs with so-called ‘cellular IRESes’ demonstrate only background activities when placed in the intercistronic position of dicistronic RNAs. In contrast, they are very active in the monocistronic context and the cap is indispensable for their activities. Surprisingly, in cultured cells or cytoplasmic extracts both the level of stimulation with the cap and the overall translation activity do not correlate with the cumulative energy of the secondary structure of the tested 5′ UTRs. The cap positive effect is still observed under profound inhibition of translation with eIF4E-BP1 but its magnitude varies for individual 5′ UTRs irrespective of the cumulative energy of their secondary structures. Thus, it is not mandatory to invoke the IRES hypothesis, at least for some mRNAs, to explain their preferential translation when eIF4E is partially inactivated.