Effects of L1-ORF2 fragments on green fluorescent protein gene expression

The retrotransposon known as long interspersed nuclear element-1 (L1) is 6 kb long, although most L1s in mammalian and other eukaryotic cells are truncated. L1 contains two open reading frames, ORF1 and ORF2, that code for an RNA-binding protein and a protein with endonuclease and reverse transcriptase activities, respectively. In this work, we examined the effects of full length L1-ORF2 and ORF2 fragments on green fluorescent protein gene (GFP) expression when inserted into the pEGFP-C1 vector downstream of GFP. All of the ORF2 fragments in sense orientation inhibited GFP expression more than when in antisense orientation, which suggests that small ORF2 fragments contribute to the distinct inhibitory effects of this ORF on gene expression. These results provide the first evidence that different 280-bp fragments have distinct effects on the termination of gene transcription, and that when inserted in the antisense direction, fragment 280-9 (the 3' end fragment of ORF2) induces premature termination of transcription that is consistent with the effect of ORF2

Identification and comparative analysis of components from the signal recognition particle in protozoa and fungi

BACKGROUND: The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the ER membrane. The SRP of metazoans is well characterized and composed of an RNA molecule and six polypeptides. The particle is organized into the S and Alu domains. The Alu domain has a translational arrest function and consists of the SRP9 and SRP14 proteins bound to the terminal regions of the SRP RNA. So far, our understanding of the SRP and its evolution in lower eukaryotes such as protozoa and yeasts has been limited. However, genome sequences of such organisms have recently become available, and we have now analyzed this information with respect to genes encoding SRP components. RESULTS: A number of SRP RNA and SRP protein genes were identified by an analysis of genomes of protozoa and fungi. The sequences and secondary structures of the Alu portion of the RNA were found to be highly variable. Furthermore, proteins SRP9/14 appeared to be absent in certain species. Comparative analysis of the SRP RNAs from different Saccharomyces species resulted in models which contain features shared between all SRP RNAs, but also a new secondary structure element in SRP RNA helix 5. Protein SRP21, previously thought to be present only in Saccharomyces, was shown to be a constituent of additional fungal genomes. Furthermore, SRP21 was found to be related to metazoan and plant SRP9, suggesting that the two proteins are functionally related. CONCLUSIONS: Analysis of a number of not previously annotated SRP components show that the SRP Alu domain is subject to a more rapid evolution than the other parts of the molecule. For instance, the RNA portion is highly variable and the protein SRP9 seems to have evolved into the SRP21 protein in fungi. In addition, we identified a secondary structure element in the Sacccharomyces RNA that has been inserted close to the Alu region. Together, these results provide important clues as to the structure, function and evolution of SRP

Identification of novel Angiogenin (ANG) gene missense variants in German patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disease characterized by the selective death of motor neurons in the motor cortex, brain stem and spinal cord. Recently, missense variants in the angiogenin gene (ANG), an angiogenic factor expressed in ventral horn motor neurons that is up-regulated by hypoxia, have been found in ALS patients of Irish/Scottish, North American, Italian, French and Dutch descent. To investigate the role of ANG in the German population, we screened for mutations by sequencing the entire coding region of the ANG gene in a large sample of 581 German ALS cases and 616 sex- and age-matched healthy controls. We identified two heterozygous missense variants, F(−13)L and K54E, in two German sporadic ALS cases but not in controls. Both missense variants are novel and have not been previously found in ALS cases. Our results suggest that missense variants in the ANG gene play a role in ALS in the German population and provide further evidence to support the hypothesis that angiogenic factors up-regulated by hypoxia are involved in the pathophysiology of ALS

Evolutionary Conservation of the Functional Modularity of Primate and Murine LINE-1 Elements

LINE-1 (L1) retroelements emerged in mammalian genomes over 80 million years ago with a few dominant subfamilies amplifying over discrete time periods that led to distinct human and mouse L1 lineages. We evaluated the functional conservation of L1 sequences by comparing retrotransposition rates of chimeric human-rodent L1 constructs to their parental L1 counterparts. Although amino acid conservation varies from ∼35% to 63% for the L1 ORF1p and ORF2p, most human and mouse L1 sequences can be functionally exchanged. Replacing either ORF1 or ORF2 to create chimeric human-mouse L1 elements did not adversely affect retrotransposition. The mouse ORF2p retains retrotransposition-competency to support both Alu and L1 mobilization when any of the domain sequences we evaluated were substituted with human counterparts. However, the substitution of portions of the mouse cys-domain into the human ORF2p reduces both L1 retrotransposition and Alu trans-mobilization by 200–1000 fold. The observed loss of ORF2p function is independent of the endonuclease or reverse transcriptase activities of ORF2p and RNA interaction required for reverse transcription. In addition, the loss of function is physically separate from the cysteine-rich motif sequence previously shown to be required for RNP formation. Our data suggest an additional role of the less characterized carboxy-terminus of the L1 ORF2 protein by demonstrating that this domain, in addition to mediating RNP interaction(s), provides an independent and required function for the retroelement amplification process. Our experiments show a functional modularity of most of the LINE sequences. However, divergent evolution of interactions within L1 has led to non-reciprocal incompatibilities between human and mouse ORF2 cys-domain sequences