Characterization and molecular targeting of CFIm25 (NUDT21/CPSF5) mRNA using miRNAs

Jan 15, 2025FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Studying and controlling CFIm25 mRNA using microRNAs

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Abstract

NUDT21 mRNA is stable and undergoes (APA) to produce different 3'UTR lengths.

CFIm25 (NUDT21) is involved in regulating RNA levels associated with neural dysfunction, fibrosis, and tumor formation.
Alternative polyadenylation allows NUDT21 mRNA to evade regulation by , which typically bind to the 3'UTR.
Three different sizes of NUDT21 mRNA were identified, each utilizing a distinct polyadenylation signal.
MicroRNAs miR-23a, miR-222, and miR-323a significantly influence NUDT21 expression levels.
These findings indicate that variations in NUDT21's 3'UTR may help regulate its expression in various physiological and pathological contexts.

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Changes in protein levels of the mammalian cleavage factor, CFIm25, play a role in regulating pathological processes including neural dysfunction, fibrosis, and tumorigenesis. However, despite these effects, little is known about how CFIm25 (NUDT21) expression is regulated at the RNA level. A potential regulator of NUDT21 mRNA are small non-coding (miRNAs). In general, miRNAs bind to the 3'untranslated regions (3'UTRs) and can target the bound mRNA for degradation or inhibit translation thus affecting the levels of protein in cells. Interestingly, a mechanism known as (APA) enables mRNAs to escape miRNA regulation by generating mRNAs with 3'UTRs of different sizes. As many miRNA target sites are located within the 3'UTR, shortening the 3'UTR allows mRNAs to evade miRNAs targeting this region. The differences in the lengths and the sequence composition of the 3'UTRs may also impact the mRNA's translatability and subcellular localization. APA has been reported to regulate over 70% of protein coding genes, thus increasing the transcript repertoire. Several proteins, including mammalian cleavage factor, CFIm25 (NUDT21), have been shown to regulate APA. In this study we wanted to determine whether CFIm25 (NUDT21), itself a regulator of APA, undergoes APA to evade miRNA regulation. We used the blood cancer mantle cell lymphoma (MCL) cells as a model and showed that in these cells, NUDT21 is relatively stable with a long half-life. In addition, the NUDT21 pre-mRNA undergoes alternative APA within the same terminal exon. The three different sized NUDT21 mRNAs have different 3'UTR lengths and they each use a different canonical polyadenylation signal, AAUAAA, for 3'end cleavage and polyadenylation. Use of miRNA mimics and inhibitors showed that miR-23a, miR-222, and miR-323a play a significant role in regulating NUDT21 expression. Hence, these results suggest that NUDT21 mRNA is stable and the different 3'UTRs generated through APA of NUDT21 play an important role in evading miRNA regulation and offers insights into how levels of CFIm25 (NUDT21) may be fine-tuned as needed under different physiological and pathological conditions.

Key numbers

70%

mRNA Stability After Actinomycin D Treatment

Percentage of NUDT21 mRNA remaining after 4 hours of treatment.

17%

Increased Luciferase Activity with miRNA Inhibitors

Percentage increase in luciferase activity after miR-222 inhibitor treatment.

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Abstract

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