Characteristics of Transfer RNA-Derived Fragments Expressed during Human Renal Cell Development: The Role of Dicer in tRF Biogenesis.

tRNA-derived fragments participate in the regulation of many processes, such as gene silencing, splicing and translation in many organisms, ranging from bacteria to humans. We were interested to know how tRF abundance changes during the different stages of renal cell development. The research model used here consisted of the following human renal cells: hESCs, HEK-293T, HK-2 and A-489 kidney tumor cells, which, together, mimic the different stages of kidney development. The characteristics of the most abundant tRFs, tRFGly(CCC), tRFVal(AAC) and tRFArg(CCU), were presented. It was found that these parental tRNAs present in cells are the source of many tRFs, thus increasing the pool of potential regulatory RNAs. Indeed, a bioinformatic analysis showed the possibility that tRFGly(CCC) and tRRFVal(AAC) could regulate the activity of a range of kidney proteins. Moreover, the distribution of tRFs and the efficiency of their expression is similar in adult and embryonic stem cells. During the formation of tRFs, HK-2 cells resemble A-498 cancer cells more than other cells. Additionally, we postulate the involvement of Dicer nuclease in the formation of tRF-5b in all the analyzed tRNAs. To confirm this, 293T NoDice cells, which in the absence of Dicer activity do not generate tRF-5b, were used.

Regulation of the p53 expression profile by hnRNP K under stress conditions.

The p53 protein is one of the transcription factors responsible for cell cycle regulation and prevention of cancer development. Its expression is regulated at the transcriptional, translational and post-translational levels. Recent years of research have shown that the 5' terminus of p53 mRNA plays an important role in this regulation. This region seems to be a docking platform for proteins involved in p53 expression, particularly under stress conditions. Here, we applied RNA-centric affinity chromatography to search for proteins that bind to the 5' terminus of p53 mRNA and thus may be able to regulate the p53 expression profile. We found heterogeneous nuclear ribonucleoprotein K, hnRNP K, to be one of the top candidates. Binding of hnRNP K to the 5'-terminal region of p53 mRNA was confirmed in vitro. We demonstrated that changes in the hnRNP K level in the cell strongly affected the p53 expression profile under various stress conditions. Downregulation or overexpression of hnRNP K caused a decrease or an increase in the p53 mRNA amount, respectively, pointing to the transcriptional mode of expression regulation. However, when hnRNP K was overexpressed under endoplasmic reticulum stress and the p53 amount has elevated no changes in the p53 mRNA level were detected suggesting translational regulation of p53 expression. Our findings have shown that hnRNP K is not only a mutual partner of p53 in the transcriptional activation of target genes under stress conditions but it also acts as a regulator of p53 expression at the transcriptional and potentially translational levels.

Translational Control in p53 Expression: The Role of 5'-Terminal Region of p53 mRNA.

In this review, the latest research concerning the structure and function of the 5'-terminal region of p53 mRNA was discussed. Special attention was focused on defined structural motifs which are present in this region, as well as their conservation and plausible functional role in translation. It is known that the length of the 5'-terminal region and the structural environment of initiation codons can strongly modulate translation initiation. The ability of this region of p53 mRNA to bind protein factors was also described with special emphasis on general principles that govern, such RNA-protein interactions. The structural alterations within the 5'-terminal region of p53 mRNA and proteins that bind to this region have a strong impact on the rate of mRNA scanning and on translation efficiency in in vitro assays, in selected cell lines, and under stress conditions. Thus, the structural features of the 5'-terminal region of p53 mRNA seem to be very important for translation and for translation regulation mechanisms. Finally, we suggested topics that, in our opinion, should be further explored for better understanding of the mechanisms of the p53 gene expression regulation at the translational level.

Translation of human Δ133p53 mRNA and its targeting by antisense oligonucleotides complementary to the 5'-terminal region of this mRNA.

The p53 protein is expressed as at least twelve protein isoforms. Within intron 4 of the human TP53 gene, a P2 transcription initiation site is located and this transcript encodes two p53 isoforms: Δ133p53 and Δ160p53. Here, the secondary structure of the 5'-terminal region of P2-initiated mRNA was characterized by means of the SHAPE and Pb2+-induced cleavage methods and for the first time, a secondary structure model of this region was proposed. Surprisingly, only Δ133p53 isoform was synthetized in vitro from the P2-initiated p53 mRNA while translation from both initiation codons occurred after the transfection of vector-encoded model mRNA to HCT116 cells. Interestingly, translation performed in the presence of the cap analogue suggested that the cap-independent process contributes to the translation of P2-initiated p53 mRNA. Subsequently, several antisense oligonucleotides targeting the 5'-terminal region of P2-initiated p53 mRNA were designed. The selected oligomers were applied in in vitro translation assays as well as in cell lines and their impact on the Δ133p53 synthesis and on cell viability was investigated. The results show that these oligomers are attractive tools in the modulation of the translation of P2-initiated p53 mRNA through attacking the 5' terminus of the transcript. Since cell proliferation is also reduced by antisense oligomers that lower the level of Δ133p53, this demonstrates an involvement of this isoform in tumorigenesis.

Variants of the 5'-terminal region of p53 mRNA influence the ribosomal scanning and translation efficiency.

The p53 protein is one of the major cell cycle regulators. The protein is expressed as at least twelve protein isoforms resulting from the use of alternative promoters, alternative splicing or downstream initiation codons. Importantly, there is growing evidence that translation initiation of p53 mRNA may be regulated by the structure and length of the naturally occurring variants of the 5'-terminal region of p53 mRNA transcripts. Here, several mRNA constructs were synthesized with variable length of the p53 5'-terminal regions and encoding luciferase reporter protein, and their translation was monitored continuously in situ in a rabbit reticulocyte lysate system. Moreover, four additional mRNA constructs were prepared. In two constructs, the structural context of AUG1 initiation codon was altered while in the other two constructs, characteristic hairpin motifs present in the p53 5'-terminal region were changed. Translation of the last two constructs was also performed in the presence of the cap analogue to test the function of the 5'-terminal region in cap-independent translation initiation. Superposition of several structural factors connected with the length of the 5'-terminal region, stable elements of the secondary structure, structural environment of the initiation codon and IRES elements greatly influenced the ribosomal scanning and translation efficiency.