Many large noncoding RNAs (lncRNAs) regulate chromatin, but the mechanisms by which they localize to genomic targets remain unexplored. We investigated the localization mechanisms of the Xist lncRNA ...during X-chromosome inactivation (XCI), a paradigm of lncRNA-mediated chromatin regulation. During the maintenance of XCI, Xist binds broadly across the X chromosome. During initiation of XCI, Xist initially transfers to distal regions across the X chromosome that are not defined by specific sequences. Instead, Xist identifies these regions by exploiting the three-dimensional conformation of the X chromosome. Xist requires its silencing domain to spread across actively transcribed regions and thereby access the entire chromosome. These findings suggest a model in which Xist coats the X chromosome by searching in three dimensions, modifying chromosome structure, and spreading to newly accessible locations.
The human genome contains thousands of long non-coding RNAs
, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen
. A specific long non-coding ...RNA-non-coding RNA activated by DNA damage (NORAD)-has recently been shown to be required for maintaining genomic stability
, but its molecular mechanism is unknown. Here we combine RNA antisense purification and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX, a component of the DNA-damage response, and contains the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex-which we term NORAD-activated ribonucleoprotein complex 1 (NARC1)-that contains the known suppressors of genomic instability topoisomerase I (TOP1), ALYREF and the PRPF19-CDC5L complex. Cells depleted for NORAD or RBMX display an increased frequency of chromosome segregation defects, reduced replication-fork velocity and altered cell-cycle progression-which represent phenotypes that are mechanistically linked to TOP1 and PRPF19-CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function, and that NORAD is required for the assembly of the previously unknown topoisomerase complex NARC1, which contributes to maintaining genomic stability. In addition, we uncover a previously unknown function for long non-coding RNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex.
Intermolecular RNA-RNA interactions are used by many noncoding RNAs (ncRNAs) to achieve their diverse functions. To identify these contacts, we developed a method based on RNA antisense purification ...to systematically map RNA-RNA interactions (RAP-RNA) and applied it to investigate two ncRNAs implicated in RNA processing: U1 small nuclear RNA, a component of the spliceosome, and Malat1, a large ncRNA that localizes to nuclear speckles. U1 and Malat1 interact with nascent transcripts through distinct targeting mechanisms. Using differential crosslinking, we confirmed that U1 directly hybridizes to 5′ splice sites and 5′ splice site motifs throughout introns and found that Malat1 interacts with pre-mRNAs indirectly through protein intermediates. Interactions with nascent pre-mRNAs cause U1 and Malat1 to localize proximally to chromatin at active genes, demonstrating that ncRNAs can use RNA-RNA interactions to target specific pre-mRNAs and genomic sites. RAP-RNA is sensitive to lower abundance RNAs as well, making it generally applicable for investigating ncRNAs.
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•A general method to identify RNA-RNA interactions for many RNAs (>80 nucleotides)•Distinguishes direct and indirect RNA-RNA interactions using different crosslinkers•U1 snRNA interacts with pre-mRNAs directly, whereas Malat1 lncRNA interacts indirectly•RNA-RNA interactions target U1 and Malat1 to chromatin at active gene loci
Comprehensive mapping of intermolecular RNA-RNA interactions for U1 snRNA and Malat1 lncRNA reveals mechanisms for targeting noncoding RNAs to chromatin at active gene loci.
The identification of HIV-1 envelope glycoprotein (Env) structures that can generate broadly neutralizing antibodies (BNAbs) is pivotal to the development of a successful vaccine against HIV-1 aimed ...at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s) that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4) receptor-bound state, thereby exposing highly conserved "CD4 induced" (CD4i) epitope(s) known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH), was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140) using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1) complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-2(7312/V434M) and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s). These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s) here, and its potential role in vaccine application.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Amino-acid sequence of squid myosin heavy chain Matulef, K; Sirokmán, K; Perreault-Micale, C L ...
Journal of muscle research and cell motility,
08/1998, Letnik:
19, Številka:
6
Journal Article
Recenzirano
This work describes the determination of the cDNA sequence encoding the myosin heavy chain (MHC) of the squid, Loligo pealei. To date, the amino-acid sequence of the MHC of calcium-regulated myosins ...is known only for two closely related species of scallops. We have determined the sequence of the entire coding region of the muscle MHC of squid, a cephalopod, and compared it with the MHC of scallops, which are pelecypods, and to other regulated and non-regulated myosins. Residues present in the MHC of only regulated myosins have been identified. The 6504 base pair (bp) sequence contains an open reading frame of 5805 nucleotides, which encodes 1935 amino acids. The sequence includes 697 bps of 3' untranslated sequence and 2 bps of 5' untranslated sequence. The deduced amino-acid sequence shows the squid MHC to be 72-73% identical and 86-87% similar to the calcium-regulated scallop MHCs cloned previously. In contrast, the squid MHC sequence is only 54-55% identical and 74% similar to skeletal MHCs of non-regulated myosins such as human fast skeletal embryonic and human perinatal skeletal muscle, and 39-40% identical and 60-62% similar to smooth muscle MHC of rabbit uterus muscle and chicken gizzard muscle, respectively. We have also detected two isoforms of the MHC in squid that appear to be spliced variants of a single myosin gene. These isoforms differ in the sequence encoding the surface loop at the nucleotide binding site. Taken together, our data may help to identify more precisely the residues that are crucial in regulated myosins.
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