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

Abstract 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

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

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

Transient expression of an adenine base editor corrects the Hutchinson-Gilford progeria syndrome mutation and improves the skin phenotype in mice

Base editing to treat diseases is progressing but tissue delivery and progenitor cells correction are challenging. Here, the authors show sustained effects and propagation of mutation-corrected progenitors by transient adenine base editor expression, improving the skin phenotype of HGPS mice. Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature ageing disorder caused by a point mutation in the LMNA gene (LMNA c.1824 C > T), resulting in the production of a detrimental protein called progerin. Adenine base editors recently emerged with a promising potential for HGPS gene therapy. However adeno-associated viral vector systems currently used in gene editing raise concerns, and the long-term effects of heterogeneous mutation correction in highly proliferative tissues like the skin are unknown. Here we use a non-integrative transient lentiviral vector system, expressing an adenine base editor to correct the HGPS mutation in the skin of HGPS mice. Transient adenine base editor expression corrected the mutation in 20.8-24.1% of the skin cells. Four weeks post delivery, the HGPS skin phenotype was improved and clusters of progerin-negative keratinocytes were detected, indicating that the mutation was corrected in both progenitor and differentiated skin cells. These results demonstrate that transient non-integrative viral vector mediated adenine base editor expression is a plausible approach for future gene-editing therapies.11Nsciescopu

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