RIP-seq reveals LINE-1 ORF1p association with p-body enriched mRNAs

Abstract

Background Long INterspersed Element-1 (LINE-1) is an autonomous retroelement able to “copy-and-paste” itself into new loci of the host genome through a process called retrotransposition. The LINE-1 bicistronic mRNA codes for two proteins, ORF1p, a nucleic acid chaperone, and ORF2p, a protein with endonuclease and reverse transcriptase activity. Both proteins bind LINE-1 mRNA in cis and are necessary for retrotransposition. While LINE-1 transcription is usually repressed in most healthy somatic cells through a plethora of mechanisms, ORF1p expression has been observed in nearly 50% of tumors, and new LINE-1 insertions have been documented in a similar fraction of tumors, including prostate cancer. Results Here, we utilized RNA ImmunoPrecipitation (RIP) and the L1EM analysis software to identify ORF1p bound RNA in prostate cancer cells. We identified LINE-1 loci that were expressed in parental androgen sensitive and androgen independent clonal derivatives. In all androgen independent cells, we found higher levels of LINE-1 RNA, as well as unique expression patterns of LINE-1 loci. Interestingly, we observed that ORF1p bound many non-LINE-1 mRNA in all prostate cancer cell lines evaluated, and polyA RNA, and RNA localized in p-bodies were especially enriched. Furthermore, the expression levels of RNAs identified in our ORF1p RIP correlated with RNAs expressed in LINE-1 positive tumors from The Cancer Genome Atlas (TCGA). Conclusion Our results show a significant remodeling of LINE-1 loci expression in androgen independent cell lines when compared to parental androgen dependent cells. Additionally, we found that ORF1p bound a significant amount of non-LINE-1 mRNA, and that the enriched ORF1p bound mRNAs are also amplified in LINE-1 expressing TCGA prostate tumors, indicating the biological relevance of our findings to prostate cancer.

Hepatitis C virus infection restricts human LINE-1 retrotransposition in hepatoma cells

“…Both proteins are required for L1 retrotransposition and assemble with their encoding L1 mRNA in cis, forming a cytoplasmic ribonucleoprotein particle (L1 RNP), that represents a retrotransposition intermediate [7,[13][14][15][16][17][18]. L1 RNPs associate with cytoplasmic stress granules and processing bodies (Pbodies) [18][19][20][21], but the role of these cellular structures in the L1 life cycle is still controversial. L1 RNPs, but also L1ORF1p alone, colocalize with various other RNA-binding proteins and for many of these proteins an RNA-dependent interaction with L1ORF1p has been reported [18,19,[21][22][23][24][25][26].…”

“…L1 RNPs associate with cytoplasmic stress granules and processing bodies (Pbodies) [18][19][20][21], but the role of these cellular structures in the L1 life cycle is still controversial. L1 RNPs, but also L1ORF1p alone, colocalize with various other RNA-binding proteins and for many of these proteins an RNA-dependent interaction with L1ORF1p has been reported [18,19,[21][22][23][24][25][26]. The retrotransposition cycle also involves trafficking of L1 RNPs into the nucleus, which requires the endosomal sorting complex required for transport (ESCRT) [27].…”

ASSESSMENT OF POTENTIAL SARS-CoV-2 VIRUS N GENE INTEGRATION INTO HUMAN GENOME REVEALS NO SIGNIFICANT IMPACT ON RT-qPCR COVID-19 DIAGNOSTIC TESTING

“…Recent experiments suggest activity of LINE-1 elements in epithelial cells [16]. In vitro experiments have shown that, in conditions of high LINE-1 derepression and expression, the machinery that mediates LINE-1 retrotransposition, can mobilize other sequences such as Alu and SVA elements, and can even lead to integration of certain cellular mRNAs, leading to the formation of processed pseudogenes [17][18][19][20]. One study even showed that LINE-1 elements can promote the low frequency integration of certain RNA viruses [21].…”

Unbiased proteomic mapping of the LINE-1 promoter using CRISPR Cas9

“…Out of these three gRNAs, we chose to use gRNA 4 and gRNA7 for our C-BERST assay. While gRNA 8 also showed high specificity to L1Hs, it did not fully align with an active L1Hs sequence that we previously identified to be expressed in LNCaP cells (Xp22.2(2)) [ 21 ]. Since Xp22.2(2) was one of the highest LINE-1 loci expressed in LNCaP cells, we decided to exclude it as a guide.…”

“…ORF1p is a nucleic acid chaperone that forms homotrimers and binds LINE-1 mRNA [ 18 , 19 ]. ORF1p has also been shown to bind ssDNA as well as non-LINE-1 mRNAs [ 20 , 21 ]. ORF2p’s endonuclease and reverse transcriptase domains provide the enzymatic activity necessary for retrotransposition [ 22 , 23 ].…”