Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA.

Abstract

P40 is the protein encoded by the first open reading frame (ORF1) of the human LINE‐1 (L1Hs) retrotransposon; it is 338 amino acids long, has a leucine zipper motif and has been found in human teratocarcinoma cell lines and some tumor cells. In this report, we describe properties of p40 in the human teratocarcinoma cell lines NTera2D1 and 2102Ep. The results indicate that: (i) most of p40 occurs in large multimeric cytoplasmic complexes, (ii) L1Hs RNA is associated with the p40 complexes, (iii) the complexes are dissociated by ribonuclease and (iv) p40 is a novel RNA‐binding protein. Cross‐linking experiments with full‐length and truncated p40 produced in Escherichia coli also showed that: (i) p40 itself can form a multimeric complex larger than 250 kDa, (ii) the leucine zipper motif and the region conserved among the predicted ORF1 polypeptides of mammalian LINE‐1s participate in complex formation and (iii) the amino terminal region is important for the stability of complex formation. Analysis of the amino acid sequence of p40 suggests that long segments of the molecule can assume an alpha‐helical configuration including the leucine zipper and the conserved region. The evidence presented here suggests that the p40 complex is a ribonucleoprotein complex containing L1Hs RNA(s) and that protein‐protein interactions in which alpha‐helix structures participate, for example coiled‐coils, may occur in the complex.

Non-LTR retrotransposons encode noncanonical RRM domains in their first open reading frame

“…For the last twenty years, NLR ORF1p proteins were studied in the absence of detailed structural information, and it was rather obscure how ORF1p would bind RNA. The present identification of RRM modules opens a new perspective and relates the observed cytoplasmic RNPs (16,19,20,(25)(26)(27) to cellular hnRNPs and mRNPs rather than to viral nucleocapsid-like RNPs. Apart from their structural role in RNP formation the RRM domains in NLR ORF1p proteins likely have specialized functions as well, assisted by accessory domains like the CTD or the CCHC zinc knuckles.…”

“…Like ORF2p, it shows a remarkable cis preference, that is, it associates preferentially with its encoding transcript (15,16). L1ORF1p can be localized in the cytoplasm (in putative stress granules) as well as in the nucleus (17,18) and can also be identified in large L1 ribonucleoprotein particles (RNPs) fractionated from cytoplasmic extracts (16,19,20).…”

L1 retrotransposition in nondividing and primary human somatic cells

“…Both L1-encoded proteins (ORF1p and ORF2p) are thought to preferentially associate with their own encoding RNA (''cis preference'') to form a ribonucleoprotein particle (RNP) (6, 7), which is a proposed retrotransposition intermediate (8,9). The L1 RNP must access the nucleus, where the L1 endonuclease cleaves genomic DNA at a degenerate consensus sequence (5Ј-TTTT͞A and variant sequences) to liberate a 3Ј hydroxyl residue that is subsequently used by the L1 reverse transcriptase (RT) as a primer to copy the L1 sequence in situ, a process termed ''targetprimed reverse transcription'' (2, 10).…”

Antisense Promoter of Human L1 Retrotransposon Drives Transcription of Adjacent Cellular Genes

“…Fulllength human L1s (L1Hs) are about 6 kb long and possess a 910-bp 5Ј untranslated region (5ЈUTR), two nonoverlapping open reading frames (ORFs), and a 205-bp 3ЈUTR. ORF1 encodes an RNA binding protein, a major component of the L1Hs ribonucleoprotein particles (15). ORF2 encodes at least two enzymatic activities, an endonuclease (11) and a reverse transcriptase (21), both of which are required for L1Hs autonomous retrotransposition (24).…”