Summary: Gene expression is dynamically regulated in a variety of mammalian physiologies. During mammalian aging, there are changes that occur in protein expression that are highly controlled by the regulatory steps in transcription, post‐transcription, and post‐translation. Although there are global profiles of human transcripts during the aging processes available, the mechanism(s) by which transcripts are differentially expressed between young and old cohorts remains unclear. Here, we report on N6‐methyladenosine (m6A) RNA modification profiles of human peripheral blood mononuclear cells (PBMCs) from young and old cohorts. An m6A RNA profile identified a decrease in overall RNA methylation during the aging process as well as the predominant modification on proteincoding mRNAs. The m6A‐modified transcripts tend to be more highly expressed than nonmodified ones. Among the many methylated mRNAs, those of DROSHA and AGO2 were heavily methylated in young PBMCs which coincided with a decreased steady‐state level of AGO2 mRNA in the old PBMC cohort. Similarly, downregulation of AGO2 in proliferating human diploid fibroblasts (HDFs) also correlated with a decrease in AGO2 mRNA modifications and steady‐state levels. In addition, the overexpression of RNA methyltransferases stabilized AGO2 mRNA but not DROSHA and DICER1 mRNA in HDFs. Moreover, the abundance of miRNAs also changed in the young and old PBMCs which are possibly due to a correlation with AGO2 expression as observed in AGO2‐depleted HDFs. Taken together, we uncovered the role of mRNA methylation on the abundance of AGO2 mRNA resulting in the repression of miRNA expression during the process of human aging.

Protein dynamics, modifications and trafficking are all processes that can modulate protein activity. Accumulating evidence strongly suggests that many proteins have distinctive roles dependent on cellular location. Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) is a transforming growth factor-β (TGF-β) superfamily protein that has a role in cancer, obesity and inflammation. NAG-1 is synthesized and cleaved into a mature peptide, which is ultimately secreted into the extracellular matrix (ECM). In this study, we have found that full-length NAG-1 is expressed in not only the cytoplasm and ECM, but also in the nucleus. NAG-1 is dynamically moved to the nucleus, exported into cytoplasm and further transported into the ECM. We have also found that nuclear NAG-1 contributes to inhibition of the Smad pathway by interrupting the Smad complex. Overall, our study indicates that NAG-1 is localized in the nucleus and provides new evidence that NAG-1 controls transcriptional regulation in the Smad pathway.

Long non-coding RNAs (lncRNAs) regulate vital biological processes, including cell proliferation, differentiation and development. A subclass of lncRNAs is synthesized from microRNA (miRNA) host genes (MIRHGs) due to pre-miRNA processing, and are categorized as miRNA-host gene lncRNAs (lnc-miRHGs). Presently, the cellular function of most lnc-miRHGs is not well understood. We demonstrate a miRNA-independent role for a nuclear-enriched lnc-miRHG in cell cycle progression. MIR100HG produces spliced and stable lncRNAs that display elevated levels during the G1 phase of the cell cycle. Depletion of MIR100HG-encoded lncRNAs in human cells results in aberrant cell cycle progression without altering the levels of miRNA encoded within MIR100HG. Notably, MIR100HG interacts with HuR/ELAVL1 as well as with several HuR-target mRNAs. Further, MIR100HG-depleted cells show reduced interaction between HuR and three of its target mRNAs, indicating that MIR100HG facilitates interaction between HuR and target mRNAs. Our studies have unearthed novel roles played by a MIRHG-encoded lncRNA in regulating RNA binding protein activity, thereby underscoring the importance of determining the function of several hundreds of lnc-miRHGs that are present in human genome.

Eukaryotic mRNA decay is tightly modulated by RNA-binding proteins (RBPs) and microRNAs (miRNAs). RBP AU-binding factor 1 (AUF1) has four isoforms resulting from alternative splicing and is critical for miRNA-mediated gene silencing with a distinct preference of target miRNAs. Previously, we have shown that AUF1 facilitates miRNA loading to Argonaute 2 (AGO2), the catalytic component of the RNA-induced silencing complex. Here, we further demonstrate that depletion of AUF1 abolishes the global interaction of miRNAs and AGO2. Single-molecule analysis revealed that AUF1 slowed down assembly of AGO2–let-7b–mRNA complex unexpectedly. However, target mRNAs recognized by both miRNA and AUF1 are less abundant upon AUF1 overexpression implying that AUF1 is a decay-promoting factor influencing multiple steps in AGO2–miRNA-mediated mRNA decay. Our findings indicate that AUF1 functions in promoting miRNA-mediated mRNA decay globally.