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Published on: June 12, 2018
Characterization and molecular targeting of CFIm25 (NUDT21/CPSF5) mRNA using miRNAs
Naazneen Khan1,2, Mahesh Gupta1, Chioniso Patience Masamha1
1Department of Pharmaceutical Sciences, Butler University, Indianapolis, Indiana, USA.
Abstract:
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 microRNAs (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 alternative polyadenylation (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.
Insights
The mammalian cleavage factor CFIm25 (NUDT21) undergoes alternative polyadenylation (APA) to generate different mRNA 3'UTRs. This APA mechanism helps NUDT21 evade microRNA regulation, fine-tuning its expression in pathological conditions.
Area of Science:
- Molecular Biology
- Gene Regulation
- Cancer Biology
Background:
- Mammalian cleavage factor, CFIm25 (NUDT21), influences neural dysfunction, fibrosis, and tumorigenesis.
- Regulation of NUDT21 expression at the RNA level, particularly by microRNAs (miRNAs), is not well understood.
- Alternative polyadenylation (APA) generates mRNAs with varying 3'UTR lengths, potentially affecting miRNA binding and regulation.
Purpose of the Study:
- To investigate whether CFIm25 (NUDT21), a known regulator of APA, itself undergoes APA.
- To determine if APA in NUDT21 mRNA serves to evade miRNA-mediated regulation.
- To explore the role of specific miRNAs in regulating NUDT21 expression.
Main Methods:
- Utilized mantle cell lymphoma (MCL) cells as a model system.
- Analyzed NUDT21 mRNA stability and half-life.
- Investigated alternative APA of NUDT21 pre-mRNA within the terminal exon.
- Employed miRNA mimics and inhibitors to assess the impact of specific miRNAs on NUDT21 expression.
Main Results:
- NUDT21 mRNA exhibits relative stability and a long half-life in MCL cells.
- NUDT21 pre-mRNA undergoes alternative APA, producing transcripts with distinct 3'UTR lengths and utilizing different polyadenylation signals.
- miR-23a, miR-222, and miR-323a were identified as significant regulators of NUDT21 expression.
Conclusions:
- NUDT21 mRNA stability is maintained, and APA generates diverse 3'UTRs that are crucial for evading miRNA regulation.
- These findings provide insights into the fine-tuning of CFIm25 (NUDT21) levels under various physiological and pathological conditions.
- The study highlights a novel regulatory mechanism for NUDT21 involving APA and miRNA interactions.
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