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Alteration of miRNome by recombinant ncRNA agents

ABSTRACT: Purpose: Identify whether recombinant ncRNAs are recognized by mammalian cells and processed into mature microRNAs, thus leading to the alteration of downstream target genes. In addition, we also explored whether cleavage is Dicer dependent. Methods: HEK293T (Dicer-WT) and 424 (Dicer-KO) cell lines were treated with 20nM of control tRNA (MSA), nCAR/miR-34a-5p, or nCAR/miR-34a-3p in triplicate. Results: Read counts of known miRNAs were analyzed using a miRDeep module, and read counts derived from nCAR/miR-34a-5p or nCAR/miR-124a-3p transfection were obtained by mapping sequence reads by an in-house developed PERL script. EdgeR was used to analyze significant (P-value < 0.05 and log2CPM > 6) differentially expressed miRNAs and sRNAs. Conclusions: Upon transfection of recombinant RNA agents in mammalian cells, nCAR/miR-34a-5p became the most dominant miRNA and was processed in a Dicer dependent manner, while nCAR/miR-124a-3p become the 7th most abundant miRNA and displayed comparable levels with or without Dicer. In both cases, different isoforms of the mature miRNA sequence were generated.

ORGANISM(S): Homo sapiens

PROVIDER: GSE103349 | GEO | 2020/09/01

REPOSITORIES: GEO

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Alteration of transcriptome by recombinant ncRNA agents

Project description:Purpose: Identify whether recombinant ncRNAs are recognized by mammalian cells and processed into mature microRNAs, thus leading to the alteration of downstream target genes. In addition, we also explored whether the alteration of known or novel target genes are Dicer dependent. Methods: HEK293T (Dicer-WT) and 424 (Dicer-KO) cell lines were treated with 20nM of control tRNA (MSA), nCAR/miR-34a-5p, or nCAR/miR-34a-3p in triplicate. Results: Read counts of known mRNAs were analyzed using a RSEM-EdgeR workflow. Sequence reads were mapped to the human reference genome (hg19) by BWA, read counts for individual genes were computed using RSEM, and EdgeR was used to analyze significant (log2FC > 1.2 or log2FC < 0.8. P-value < 0.05 and log2CPM > 5) differentially expressed mRNAs. Conclusions: Upon transfection of recombinant RNA agents in mammalian cells, known and novel target genes were regulated in a Dicer dependent manner (nCAR/miR-34a-5p) and also in a Dicer independent manner (nCAR/miR-124a-3p).

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Identification of SART3 as a pre-miR-34a–binding protein

Project description:MicroRNAs (miRNAs or miRs) are small, noncoding RNAs that are implicated in the regulation of nearly all biological processes. Global miRNA biogenesis is altered in many cancers and RNA-binding proteins (RBPs) have been shown to play a role in this process, presenting a promising avenue for targeting miRNA dysregulation in disease. miR-34a exhibits tumor-suppressive functions by targeting cell cycle regulators CDK4/6 and anti-apoptotic factor Bcl-2, among other regulatory pathways such as Wnt, TGF-, and Notch signaling. Many cancers show downregulation or loss of miR-34a, and synthetic miR-34a supplementation has been shown to inhibit tumor growth in vivo; however, the post-transcriptional mechanisms by which miR-34a is lost in cancer are not entirely understood. Here, we have used a proteomics-mediated approach to identify Squamous cell carcinoma antigen recognized by T-cells 3 (SART3) as a putative pre-miR-34a-binding protein. SART3 is a spliceosome recycling factor and nuclear RBP with no previously reported role in miRNA regulation. We demonstrate that SART3 binds pre-miR-34a with specificity over pre-let-7d and begin to elucidate a new functional role for this protein in non-small lung cancer cells. Overexpression of SART3 led to increased miR-34a levels, downregulation of the miR-34a target genes CDK4 and CDK6, and cell cycle arrest in the G1 phase. In vitro binding studies showed that the RNA-recognition motifs within the SART3 sequence are responsible for selective pre-miR-34a binding. Collectively, our results present evidence for an influential role of SART3 in miR-34a biogenesis and cell cycle progression.

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