An Exportin-1–dependent microRNA biogenesis pathway during human cell quiescence

Abstract

The reversible state of proliferative arrest known as "cellular quiescence" plays an important role in tissue homeostasis and stem cell biology. By analyzing the expression of miRNAs and miRNAprocessing factors during quiescence in primary human fibroblasts, we identified a group of miRNAs that are induced during quiescence despite markedly reduced expression of Exportin-5, a protein required for canonical miRNA biogenesis. The biogenesis of these quiescence-induced miRNAs is independent of Exportin-5 and depends instead on Exportin-1. Moreover, these quiescence-induced primary miRNAs (pri-miRNAs) are modified with a 2,2,7-trimethylguanosine (TMG)-cap, which is known to bind Exportin-1, and knockdown of Exportin-1 or trimethylguanosine synthase 1, responsible for (TMG)-capping, inhibits their biogenesis. Surprisingly, in quiescent cells Exportin-1-dependent pri-miR-34a is present in the cytoplasm together with a small isoform of Drosha, implying the existence of a different miRNA processing pathway in these cells. Our findings suggest that during quiescence the canonical miRNA biogenesis pathway is down-regulated and specific miRNAs are generated by an alternative pathway to regulate genes involved in cellular growth arrest.M ost metazoan cells enter a reversible cell-cycle arrest known as "cellular quiescence" when they are exposed to antimitogenic signals or an environment unfavorable for proliferation (1, 2). In mammalian cells, quiescence (also known as "G 0 arrest") is characterized by reduced DNA replication, altered metabolism, increased autophagy, and increased expression of cyclin-dependent kinase inhibitors such as p27 Kip1 (3,4). In vitro, quiescence can be induced in primary cells by serum starvation, contact inhibition, and loss of adhesion to a substrate (5). Quiescence is involved in important cellular processes, including the balance between differentiation and self-renewal in different types of stem cells, and dysregulation of quiescence could favor carcinogenesis. However, the molecular mechanisms that regulate quiescence are poorly understood (6). miRNAs are small, noncoding RNAs ∼22-nt long that regulate the expression of protein-coding genes by base-pairing with the 3′ UTR of mRNAs, repressing the translation and/or inducing the degradation of the target mRNA (7,8). In canonical miRNA biogenesis in mammalian cells, miRNAs are transcribed by RNA polymerase II to produce 7-methylguanosine (m 7 G)-capped primary miRNAs (pri-miRNAs) containing one or more bulged hairpin structures that are recognized by the nuclear microprocessor, which consists of the RNase III enzyme Drosha and the dsRNA-binding protein DGCR8 (DiGeorge syndrome critical region gene 8) (9-12). Specific cleavage of the pri-miRNA by the nuclear microprocessor generates an ∼70-nt stem-loop structure known as the "precursor miRNA" (pre-miRNA), which is recognized and transported to the cytoplasm by 10,11,13). Subsequently, pre-miRNA is cleaved by the cytoplasmic RNase III enzyme Dicer (14-17), generating a double-stranded mature mi...

Immunoprecipitation of Tri-methylated Capped RNA

“…The recovery of (TMG)-capped RNAs was measured by RT-PCR amplification of well-known hypermethylated RNAs such as small nuclear RNA (snRNA) U7 (Figure 2) or small nucleolar RNA (snoRNA) U3 (Martinez et al ., 2017). …”

“…Previous publications have shown the ability to pull-down (TMG)-cap RNAs, such as snRNAs and snoRNAs, with specific antibodies against (TMG)-cap RNAs (Luhrmann et al ., 1982). Our previous publications demonstrated for the first time the pull-down of (TMG)-cap pri-miRNAs in human cells (Martinez et al , 2017). Understanding which RNAs could be modified with a TMG-cap will provide new important insights into RNA biogenesis in normal or disease-related conditions.…”

XPO5 promotes primary miRNA processing independently of RanGTP

“…Functional studies showed a requirement of XPO5 to transport pre-miRNAs from the nucleus to the cytoplasm [2][3][4] , providing the evidence that XPO5 is an essential component of miRNA biogenesis 7 . However, XPO5-independent pre-miRNA export pathway was also reported 8,9 . Notably, 7methylguanosine-capped pre-miRNAs whose biogenesis is independent of the DROSHA/DGCR8 microprocessor are exported via the PHAX-XPO1 pathway 9 .…”

“…Incubation of increasing amount of XPO5 leads to increased processing efficiency of pri-mir-19a (lanes 3-6). Incubation of increasing amount of the XPO5 mutant also results in increased processing efficiency of pri-mir-19a (lanes[8][9][10][11]. b Preincubation of XPO5 and XPO5 mutant enhances the microprocessor cleavage of truncated pri-mir-19a v1.…”

Nuclear export mechanisms of circular RNAs: size does matter

“…Small non-coding RNA (microRNA) maturation occurs in the cytosol and requires efficient nuclear export through the karyopherin family member exportin 5 (XPO5) (9). A limited set of studies have also shown that aside from XPO5, the export of few precursor miRNAs occurs through XPO1 as well (10). The mRNAs have a distinct export mechanism that occurs with the help of adaptor serine/arginine rich proteins that promote the recruitment of heterodimeric mRNA export receptor NXF1-NXT1 which facilitates exit through the nuclear pore (11).…”