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Ribonuclease activity and RNA binding of recombinant human Dicer.

ABSTRACT: RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. Here, we report cloning and expression of the 218 kDa human Dicer, and characterization of its ribonuclease activity and dsRNA-binding properties. The recombinant enzyme generated approximately 21-23 nucleotide products from dsRNA. Processing of the microRNA let-7 precursor by Dicer produced an apparently mature let-7 RNA. Mg(2+) was required for dsRNase activity, but not for dsRNA binding, thereby uncoupling these reaction steps. ATP was dispensable for dsRNase activity in vitro. The Dicer.dsRNA complex formed at high KCl concentrations was catalytically inactive, suggesting that ionic interactions are involved in dsRNA cleavage. The putative dsRNA-binding domain located at the C-terminus of Dicer was demonstrated to bind dsRNA in vitro. Human Dicer expressed in mammalian cells colocalized with calreticulin, a resident protein of the endoplasmic reticulum. Availability of the recombinant Dicer protein will help improve our understanding of RNA silencing and other Dicer-related processes.

SUBMITTER: Provost P

PROVIDER: S-EPMC131075 | biostudies-other | 2002 Nov

REPOSITORIES: biostudies-other

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Ribonuclease activity and RNA binding of recombinant human Dicer.

Provost Patrick P Dishart David D Doucet Johanne J Frendewey David D Samuelsson Bengt B Rådmark Olof O

The EMBO journal 20021101 21

RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. Here, we report cloning and expression of the 218 kDa human Dicer, and characterization of its ribonuclease activity and dsRNA-binding properties. The recombinant enzyme generated approximately 21-23 nucleotide products from dsRNA. Processing of the microRNA ...[more]

PMID: 12411504

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Project description:Gene expression dictates fundamental cellular processes and its de-regulation leads to pathological conditions. A key contributor to the fine-tuning of gene expression is Dicer, an RNA-binding protein (RBPs) that forms complexes and affects transcription by acting at the post-transcriptional level via the targeting of mRNAs by Dicer-produced small non-coding RNAs. This review aims to present the contribution of Dicer protein in a wide spectrum of human pathological conditions, including cancer, neurological, autoimmune, reproductive and cardiovascular diseases, as well as viral infections. Germline mutations of Dicer have been linked to Dicer1 syndrome, a rare genetic disorder that predisposes to the development of both benign and malignant tumors, but the exact correlation of Dicer protein expression within the different cancer types is unclear, and there are contradictions in the data. Downregulation of Dicer is related to Geographic atrophy (GA), a severe eye-disease that is a leading cause of blindness in industrialized countries, as well as to psychiatric and neurological diseases such as depression and Parkinson's disease, respectively. Both loss and upregulation of Dicer protein expression is implicated in severe autoimmune disorders, including psoriasis, ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis and autoimmune thyroid diseases. Loss of Dicer contributes to cardiovascular diseases and causes defective germ cell differentiation and reproductive system abnormalities in both sexes. Dicer can also act as a strong antiviral with a crucial role in RNA-based antiviral immunity. In conclusion, Dicer is an essential enzyme for the maintenance of physiology due to its pivotal role in several cellular processes, and its loss or aberrant expression contributes to the development of severe human diseases. Further exploitation is required for the development of novel, more effective Dicer-based diagnostic and therapeutic strategies, with the goal of new clinical benefits and better quality of life for patients.

Dataset Information

Ribonuclease activity and RNA binding of recombinant human Dicer.

Publications

Ribonuclease activity and RNA binding of recombinant human Dicer.

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