Translation of the human LINE-1 element, L1Hs.

McMillan, J.; Singer, Maxine

doi:10.1073/pnas.90.24.11533

Cited by 70 publications

(59 citation statements)

References 31 publications

(41 reference statements)

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“…Some non-LTR retrotransposons, like human L1 or I factor of Drosophila, have two separate ORFs. In these elements, internal translation initiation was indicated because there was no detection of ORF1-ORF2 fusion proteins (5,12,27). Many non-LTR retrotransposons do not have overlapping stop-start codons, but most of them encode methionines at the beginning of endonuclease domains (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Eukaryotic Translational Coupling in UAAUG Stop-Start Codons for the Bicistronic RNA Translation of the Non-Long Terminal Repeat Retrotransposon SART1

Kojima

Matsumoto

Fujiwara

2005

Molecular and Cellular Biology

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Most eukaryotic cellular mRNAs are monocistronic; however, many retroviruses and long terminal repeat (LTR) retrotransposons encode multiple proteins on a single RNA transcript using ribosomal frameshifting. Non-long terminal repeat (non-LTR) retrotransposons are considered the ancestor of LTR retrotransposons and retroviruses, but their translational mechanism of bicistronic RNA remains unknown. We used a baculovirus expression system to produce a large amount of the bicistronic RNA of SART1, a non-LTR retrotransposon of the silkworm, and were able to detect the second open reading frame protein (ORF2) by Western blotting. The ORF2 protein was translated as an independent protein, not as an ORF1-ORF2 fusion protein.We revealed by mutagenesis that the UAAUG overlapping stop-start codon and the downstream RNA secondary structure are necessary for efficient ORF2 translation. Increasing the distance between the ORF1 stop codon and the ORF2 start codon decreased translation efficiency. These results are different from the eukaryotic translation reinitiation mechanism represented by the yeast GCN4 gene, in which the probability of reinitiation increases as the distance between the two ORFs increases. The translational mechanism of SART1 ORF2 is analogous to translational coupling observed in prokaryotes and viruses. Our results indicate that translational coupling is a general mechanism for bicistronic RNA translation.In many retroviruses and retrotransposons, gag protein and pol protein are encoded in different open reading frames (ORFs) on a single RNA transcript (4, 14). They translate these two proteins as gag-pol polyproteins. Retroviruses, such as human immunodeficiency virus type 1 and human T-cell lymphotropic virus type 1, have a Ϫ1 translational frameshift signal, and Ty1, one of the yeast long terminal repeat (LTR) retrotransposons, has a ϩ1 frameshift signal to translate gagpol polyproteins (4, 14). The signal for retroviral Ϫ1 frameshifting in RNA is composed of two structures, a slippery sequence where the frameshifting takes place and the downstream stem-loop or pseudoknot structure (16). The general slippery sequence in retroviruses is the heptamer nucleotide sequence X XXY YYZ. The tRNA initially bound to XXY slips to bind to XXX at the P site, and the tRNA initially bound to YYZ slips to bind to YYY at the A site. The mechanism for ϩ1 frameshifting in Ty1 is entirely different from the retroviral Ϫ1 frameshifting. A codon for low available tRNA interrupts the translation elongation and induces the tRNA slippage (15).Non-LTR retrotransposons are considered the ancestor of LTR retrotransposons and retroviruses (25). Although more ancient classes of non-LTR retrotransposons have a single ORF, the recently branched non-LTR retrotransposons usually have two ORFs (24). The first ORF (ORF1) of the latter type of non-LTR retrotransposons encodes a gag-like protein and the second ORF (ORF2) a pol-like protein, which are similar to LTR retrotransposons and retroviruses. ORF2 encodes two essential catalytic do...

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Section: Discussionmentioning

confidence: 99%

Eukaryotic Translational Coupling in UAAUG Stop-Start Codons for the Bicistronic RNA Translation of the Non-Long Terminal Repeat Retrotransposon SART1

Kojima

Matsumoto

Fujiwara

2005

Molecular and Cellular Biology

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“…To investigate the hypothesis that the HIV-1 gag ORF exhibits IRES activity, we undertook a series of experiments involving gag expression in poliovirus-infected cells. Because poliovirus mediates a rapid inhibition of cap-dependent translation in permissive host cells (23,56), it has been widely used in the investigation of the cap dependency of translation of various mRNAs. (Fig.…”

Section: Expression Of Pr55mentioning

confidence: 99%

The Human Immunodeficiency Virus Type 1gagGene Encodes an Internal Ribosome Entry Site

Buck

Shen²,

Egan³

et al. 2001

J Virol

148

161

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Several retroviruses have recently been shown to promote translation of their gag gene products by internal ribosome entry. In this report, we show that mRNAs containing the human immunodeficiency virus type 1 (HIV-1) gag open reading frame (ORF) exhibit internal ribosome entry site (IRES) activity that can promote translational initiation of Pr55 gag . Remarkably, this IRES activity is driven by sequences within the gag ORF itself and is not dependent on the native gag 5-untranslated region (UTR). This cap-independent mechanism for Pr55 gag translation may help explain the high levels of translation of this protein in the face of major RNA structural barriers to scanning ribosomes found in the gag 5 UTR. The gag IRES activity described here also drives translation of a novel 40-kDa Gag isoform through translational initiation at an internal AUG codon found near the amino terminus of the Pr55 gag capsid domain. Our findings suggest that this low-abundance Gag isoform may be important for wild-type replication of HIV-1 in cultured cells. The activities of the HIV-1 gag IRES may be an important feature of the HIV-1 life cycle and could serve as a novel target for antiretroviral therapeutic strategies.Like other complex retroviruses, human immunodeficiency virus type 1 (HIV-1) employs a variety of mechanisms to express numerous proteins from a single-genomic-length RNA transcript. One such mechanism is the regulated splicing of the primary transcript into more than 30 distinct mRNA species (65,(74)(75)(76). Other mechanisms for the production of different polypeptides from mature viral mRNA species rely on translational events that are atypical of normal host protein translation.The initiation of translation of most eukaryotic mRNAs is thought to occur by a process involving ribosome scanning. In this model, the 40S ribosomal subunit binds to the 5Ј end of a capped mRNA (80, 81), commences scanning toward the 3Ј end, and initiates translation upon encountering an AUG codon in suitable context (often referred to as a "Kozak" context) (47,49,50). The 60S ribosomal subunit is then recruited, and translation of a single polypeptide begins.Translation of some HIV-1 mRNAs can depart from this basic model in several ways. Translation of the pol open reading frame (ORF) requires a Ϫ1 frameshift at a "slippery" sequence within the gag ORF. This frameshift allows roughly 5 to 10% of translating ribosomes to bypass the normal gag stop codon, enabling translation of a Gag-Pol precursor protein (reviewed in reference 32). Other HIV-1 gene products, including Env and Nef, have been shown to be translated by "leaky" scanning of ribosomes past the vpu and rev AUGs, respectively (74,75,77).Another alternative mechanism for translational initiation is driven by RNA structural elements referred to as internal ribosome entry sites (or segments) (IRESs). IRESs are thought to promote initiation of translation by directly binding ribosomes, with participation of other host cell factors, in a manner independent of the mRNA cap or of scann...

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“…Upon translation, ORF1p and ORF2p exhibit cis-preference and bind their encoding mRNA (30, 32) to form a ribonucleoprotein particle (RNP) (7,16,45,46). Although ORF1p is relatively abundant in cytoplasmic RNPs (7, 45, 46), the unconventional translation mechanism of human L1 ORF2 likely gives rise to fewer molecules of ORF2p per L1 mRNA (16,47,48). The L1 RNP translocates into the nucleus by a process that can occur in the absence of nuclear envelope breakdown (49).…”

mentioning

confidence: 99%

Similarities between long interspersed element-1 (LINE-1) reverse transcriptase and telomerase

Kopera¹,

Moldovan²,

Morrish³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Long interspersed element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that contain activities required for conventional retrotransposition by a mechanism termed target-site primed reverse transcription. Previous experiments in XRCC4 or DNA protein kinase catalytic subunit-deficient CHO cell lines, which are defective for the nonhomologous endjoining DNA repair pathway, revealed an alternative endonuclease-independent (ENi) pathway for L1 retrotransposition. Interestingly, some ENi retrotransposition events in DNA protein kinase catalytic subunit-deficient cells are targeted to dysfunctional telomeres. Here we used an in vitro assay to detect L1 reverse transcriptase activity to demonstrate that wild-type or endonuclease-defective L1 ribonucleoprotein particles can use oligonucleotide adapters that mimic telomeric ends as primers to initiate the reverse transcription of L1 mRNA. Importantly, these ribonucleoprotein particles also contain a nuclease activity that can process the oligonucleotide adapters before the initiation of reverse transcription. Finally, we demonstrate that ORF1p is not strictly required for ENi retrotransposition at dysfunctional telomeres. Thus, these data further highlight similarities between the mechanism of ENi L1 retrotransposition and telomerase.L ong interspersed element-1 sequences (LINE-1s or L1s) are abundant non-long terminal repeat (non-LTR) retrotransposons that constitute approximately one-sixth (i.e., ≈17%) of human nuclear DNA (1). An estimated 80-100 full-length, retrotransposition competent L1s (RC-L1s) are present in a typical diploid human genome, and a small number, termed "hot L1s" exhibit high retrotransposition efficiencies in cultured human cells (2). Human RC-L1s are ≈6 kb (3, 4) and encode two proteins (ORF1p and ORF2p) required for retrotransposition (5). ORF1p is a 40-kDa protein (6) with RNA binding (7-12) and nucleic acid chaperone activities (13-15). ORF2p is a 150-kDa protein (16, 17) with endonuclease (EN) (18,19) and reverse transcriptase (RT) (20, 21) activities.The mobilization of RC-L1s can be mutagenic (22); germ line and, less often, somatic L1 insertions have led to ≈65 sporadic cases of disease in humans (reviewed in ref. 23). ORF1p and/or ORF2p also can act in trans to mobilize short interspersed elements [e.g., Alu (24) and SINE-VNTR-Alu (25) elements], certain noncoding RNAs [e.g., U6 snRNA and small nucleolar RNAs (24, 26-29)], and some cellular mRNAs, which leads to the formation of processed pseudogenes (30-32). In total, these trans retrotransposition events account for at least an additional 10% of human DNA (1). Moreover, several recent studies have further demonstrated that L1-mediated retrotransposition events are responsible for a significant proportion of interindividual genetic variation in the human population (33-39) and may cause intraindividual variation in the mammalian nervous system (40, 41). L1 retrotransposition likely occurs by a mechanism termed target-site primed reverse transcription (TPRT) (42)...

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Translation of the human LINE-1 element, L1Hs.

Cited by 70 publications

References 31 publications

Eukaryotic Translational Coupling in UAAUG Stop-Start Codons for the Bicistronic RNA Translation of the Non-Long Terminal Repeat Retrotransposon SART1

Eukaryotic Translational Coupling in UAAUG Stop-Start Codons for the Bicistronic RNA Translation of the Non-Long Terminal Repeat Retrotransposon SART1

The Human Immunodeficiency Virus Type 1gagGene Encodes an Internal Ribosome Entry Site

Similarities between long interspersed element-1 (LINE-1) reverse transcriptase and telomerase

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