Developmental change in subcellular location of Bp-1 protein with an ability to interact with both identifier sequence and its brain-specific transcript, BC-1 RNA.

Identifier sequences are transcribed to generate a brain-specific BC-1 RNA present as a ribonucleoprotein particle in the dendrites and somata of neurons. This ribonucleoprotein particle contains an identifier sequence-binding protein (Bp-1 protein). We report here the purification of BC-1 RNA and demonstrate that Bp-1 protein interacts directly with the RNA. We also demonstrate an accumulation of Bp-1 protein in the nucleus of brain cells from mouse fetus and newborns that precedes the postnatal increase in BC-1 RNA. Cytoplasmic Bp-1 protein present in a complex with BC-1 RNA increases postnatally with a concomitant decrease in nuclear Bp-1 protein. These observations suggest that Bp-1 protein may play a role(s) in the synthesis and nuclear export of BC-1 RNA.     

LINE-1编码的逆转录酶在肿瘤形成过程中的作用

逆转录转座子LINE-1是人类基因组中最大的转座子家族,约有500 000个拷贝,占人类基因组总量的17%,同时它也是人类基因组中唯一具有自主转座能力的转座子。LINE-1编码的逆转录酶是LINE-1转座所必需,近年来的研究表明该酶在包括肿瘤形成等重要的病理或生理过程中都发挥着重要的作用。抑制该酶的活性可阻滞肿瘤发展的进程、恢复肿瘤细胞的分化状态以及改变肿瘤细胞的转录组谱。本文根据近年来对LINE-1编码的逆转录酶的研究进展,介绍该酶通过对非编码RNA转录组谱的调控在肿瘤形成过程中产生的作用,以期为肿瘤的早期诊断以及抗肿瘤药物的开发提供一些线索。     

Spatial assembly and RNA binding stoichiometry of a LINE-1 protein essential for retrotransposition

LINE-1, or L1, is a highly successful retrotransposon in mammals, comprising 17% and 19% of the human and mouse genomes, respectively. L1 retrotransposition and hence amplification requires the protein products of its two open reading frames, ORF1 and ORF2. The sequence of the ORF1 protein (ORF1p) is not related to any protein with known function. ORF1p has RNA binding and nucleic acid chaperone activities that are both required for retrotransposition. Earlier studies have shown that ORF1p forms a homotrimer with an asymmetric dumbbell shape, in which a rod separates a large end from a small end. Here, we determine the topological arrangement of monomers within the homotrimer by comparing atomic force microscopy (AFM) images of the full ORF1p with those of truncations containing just the N or C-terminal regions. In addition, AFM images of ORF1p bound to RNA at high protein/RNA molar ratios show that ORF1p can form tightly packed clusters on RNA, with binding occurring at the C-terminal domain. The number of bound ORF1p trimers increases with increasing length of the RNA, revealing that the binding site size is about 50 nt, a value confirmed by nitrocellulose filter binding under stoichiometric conditions. These results are consistent with a role for ORF1p during L1 retrotransposition that includes both coating the RNA and acting as a nucleic acid chaperone. Furthermore, these in vitro L1 ribonucleoprotein particles provide insight into the structure of the L1 retrotransposition intermediate.     

Visualizing induced fit in early assembly of the human signal recognition particle

Assembly of almost all ribonucleoprotein complexes involves induced fit in the RNA and, thus, formation of one or more intermediate states. In assembly of the human signal recognition particle (SRP), we show that SRP19 binding to SRP RNA involves obligatory intermediates. An apparent discrepancy exists between the ratio of dissociation and association rate constants, determined in a partitioning experiment, and the equilibrium binding constant; this kinetic signature reflects formation of a stable intermediate in assembly of the ribonucleoprotein complex. Assembly intermediates were observed directly by time-resolved footprinting. SRP19 binds rapidly to SRP RNA to form an initial labile, but structurally specific, encounter complex involving both helices III and IV. Two subsequent steps of structural consolidation yield the native RNA-protein interface. SRP19 binding stabilizes helix IV in the region recognized by SRP54, consistent with protein-protein cooperativity mediated in part by mutual recognition of similar RNA structures. This mechanism illustrates principles general to ribonucleoprotein assembly reactions that rely on recruitment of architectural RNA binding proteins.     

Characterization of the prosome from Drosophila and its similarity to the cytoplasmic structures formed by the low molecular weight heat-shock proteins.

We have identified and characterized a ribonucleoprotein structure from the cytoplasm of Drosophila melanogaster tissue culture cells which is equivalent to the prosome, a recently described ribonucleoprotein particle of duck and mouse cells. During the recovery period following heat shock, the low mol. wt. heat-shock proteins form cytoplasmic ribonucleoprotein particles which co-purify with the Drosophila prosome. Both ribonucleoprotein particles share several structural properties but their protein constituents differ in their metabolism and cellular localization during the heat treatment. We also report the partial nucleotide sequences of several small RNA species associated with the Drosophila prosome. One of them has a strong sequence homology with the U6 mammalian small nuclear RNA.     

ERCC1/XPF limits L1 retrotransposition

Retrotransposons are currently active in the human and mouse genomes contributing to novel disease mutations and genomic variation via de novo insertions. However, little is known about the interactions of non-long terminal repeat (non-LTR) retrotransposons with the host DNA repair machinery. Based on the model of retrotransposition for the human and mouse LINE-1 element, one likely intermediate is an extension of cDNA that is heterologous to the genomic target, a flap intermediate. To determine whether a human flap endonuclease could recognize and process this potential intermediate, the genetic requirement for the ERCC1/XPF heterodimer during LINE-1 retrotransposition was characterized. Reduction of XPF in human cells increased retrotransposition whereas complementation of ERCC1-deficiency in hamster cells reduced retrotransposition. These results demonstrate for the first time that DNA repair enzymes act to limit non-LTR retrotransposition and may provide insight into the genetic instability phenotypes of ercc1 and xpf individuals.     

Ribonucleoprotein particle appearing during sporulation in yeast.

During sporulation of Saccharomyces cerevisiae, most strains accumulate an unmethylated 20S RNA. Contrary to previous reports, this sporulation 20S RNA is distinct from the short-lived methylated 20S RNA precursor of 18S rRNA. This RNA species was found in a cytoplasmic 32S ribonucleoprotein particle consisting of one single-stranded 20S RNA molecule and 18 to 20 identical protein subunits of molecular weight 23,000. The ribonucleoprotein particle was resistant to ribonuclease digestion, although purified 20S RNA was ribonuclease sensitive. Both the RNA and the protein of the 32S ribonucleoprotein particle were only synthesized under conditions that induce sporulation. The accumulation of 20S RNA depended on continued protein synthesis but was actinomycin D insensitive, despite a high guanine-plus-cytosine content. Synthesis of 20S RNA stopped when cells were removed from sporulation conditions and placed in growth medium.     

Computational and biological inference of gene regulatory networks of the LINE-1 retrotransposon

Computational approaches were used to define structural and functional determinants of a putative genetic regulatory network of murine LINE-1 (long interspersed nuclear element-1), an active mammalian retrotransposon that uses RNA intermediates to populate new sites throughout the genome. Polymerase (RNA) II polypeptide E AI845735 and mouse DNA homologous to Drosophila per fragment M12039 were identified as primary attractors. siRNA knockdown of the aryl hydrocarbon receptor NM_013464 modulated gene expression within the network, including LINE-1, Sgpl1, Sdcbp, and Mgst1. Genes within the network did not exhibit physical proximity and instead were dispersed throughout the genome. The potential impact of individual members of the network on the global dynamical behavior of LINE-1 was examined from a theoretical and empirical framework.     

Non-polyadenylated 22 s ribonucleoprotein particle is insensitive to translational inhibitor RNA of cryptobiotic gastrulae of Artemia salina.

A free cytoplasmic 22 S ribonucleoprotein particle exhibiting a major template activity in rabbit reticulocyte system has been identified in the cryptobiotic gastrulae of Artemia salina. This particle contains non-polyadenylated 9 S messenger RNA which codes primarily for a non-histone basic protein with an apparent molecular weight of 26 000 daltons. We have previously demonstrated the presence of a translational inhibitor RNA which is apparently responsible for transforming polyadenylated messenger (Slegers et al., FEBS Letters 80, 390-394, 1977). This inhibitor RNA was found to be completely ineffective on the template activity of non-polyadenylated 22 S messenger ribonucleoprotein, confirming the specificity of this regulatory RNA for polyadenylate sequences.