Abstract
MCPIP1, also known as Regnase-1, is a ribonuclease crucial for regulation of stability of transcripts related to inflammatory processes. Here, we report that MCPIP1 acts as an endonuclease by degrading several stem-loop RNA structures and single-stranded RNAs. Our studies revealed cleavage sites present in the stem-loops derived from the 3′ untranslated region of the interleukin-6 transcript. Furthermore, MCPIP1 induced endonuclease cleavage at the loop motif of stem-loop structures. Additionally, we observed that MCPIP1 could cleave single-stranded RNA fragments. However, RNA substrates shorter than 6 nucleotides were not further affected by MCPIP1 nucleolytic activity. In this study, we also determined the dissociation constants of full-length MCPIP1D141N and its ribonuclease domain PIN D141N with twelve oligonucleotides substrates. The equilibrium binding constants (Kd) for MCPIP1D141N and the RNA targets were approximately 10 nM. Interestingly, we observed that the presence of a zinc finger in the PIN domain increases the affinity of this protein fragment to 25-nucleotide-long stem-loop RNA but not to shorter ones. Furthermore, size exclusion chromatography of the MCPIP1 and PIN proteins suggested that MCPIP1 undergoes homooligomerization during interaction with RNA substrates. Our results provide insight into the mechanism of MCPIP1 substrate recognition and its affinity towards various oligonucleotides.
Highlights
Ribonuclease degradation of mRNA is an essential mechanism to control the level of selected transcripts in cells
Previous studies indicated that MCPIP1 is a selective ribonuclease that cleaves translationally active mRNA at the 3′UTR5
We hypothesized that the region of MCPIP1 that is crucial for RNA binding must be proximal to the catalytic cleft in the PIN domain since the 7-nt-long mIL-682–88 substrate was still bound with high affinity to the PIND141N domain
Summary
Ribonuclease degradation of mRNA is an essential mechanism to control the level of selected transcripts in cells. PIN domains are commonly present in various eukaryotic and prokaryotic nucleases that cleave different classes of RNA molecules, including mRNA, rRNA, tRNA and viral RNAs12,13. The crystal structure of the PIN domain revealed the positively charged loop sequence that is located near the catalytic core of MCPIP1. This loop may mediate the interaction with negatively charged phosphate groups of oligonucleotide backbones[11]. Homooligomerization of MCPIP1 occurs through the C-terminal domain, which is enriched in proline residues Deletion of this region decreased ribonucleolytic activity of MCPIP120. 25 nt 25 nt 25 nt 25 nt 17 nt 18 nt 17 nt 12 nt 7 nt 12 nt 25 nt 22 nt 12 nt 45 nt 12 nt 12 bp catalytic site (D141N) retained the ability to recognize RNA, and formation of the nucleoprotein complex was observed in gel shift electromobility assays[19,21]
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