scholarly journals Biochemical and Structural Insights into RNA Binding by Ssh10b, a Member of the Highly Conserved Sac10b Protein Family in Archaea

2013 ◽  
Vol 289 (3) ◽  
pp. 1478-1490 ◽  
Author(s):  
Li Guo ◽  
Jingjin Ding ◽  
Rong Guo ◽  
Yanjie Hou ◽  
Da-Cheng Wang ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Rna Secondary Structure ◽  
Rna Binding ◽  
Amino Acid Residues ◽  
Sulfolobus Shibatae ◽  
Β Sheet ◽  
Structural Insights ◽  
Dsdna Binding ◽  
Local Distortions

Proteins of the Sac10b family are highly conserved in Archaea. Ssh10b, a member of the Sac10b family from the hyperthermophilic crenarchaeon Sulfolobus shibatae, binds to RNA in vivo. Here we show that binding by Ssh10b destabilizes RNA secondary structure. Structural analysis of Ssh10b in complex with a 25-bp RNA duplex containing local distortions reveals that Ssh10b binds the two RNA strands symmetrically as a tetramer with each dimer bound asymmetrically to a single RNA strand. Amino acid residues involved in double-stranded RNA binding are similar, but non-identical, to those in dsDNA binding. The dimer-dimer interaction mediated by the intermolecular β-sheet appears to facilitate the destabilization of base pairing in the secondary structure of RNA. Our results suggest that proteins of the Sac10b family may play important roles in RNA transactions requiring destabilization of RNA secondary structure in Sulfolobus.

scholarly journals Amount of RNA secondary structure required to induce an alternative splice.

1987 ◽  
Vol 7 (9) ◽  
pp. 3194-3198 ◽  
Author(s):  
D Solnick ◽  
S I Lee
Keyword(s):  
Alternative Splicing ◽  
Secondary Structure ◽  
Alternative Splice ◽  
Rna Secondary Structure ◽  
Secondary Structures ◽  
Splice Sites ◽  
Perfect Complementarity ◽  
Set Up

We set up an alternative splicing system in vitro in which the relative amounts of two spliced RNAs, one containing and the other lacking a particular exon, were directly proportional to the length of an inverted repeat inserted into the flanking introns. We then used the system to measure the effect of intramolecular complementarity on alternative splicing in vivo. We found that an alternative splice was induced in vivo only when the introns contained more than approximately 50 nucleotides of perfect complementarity, that is, only when the secondary structure was much more stable than most if not all possible secondary structures in natural mRNA precursors. We showed further that intron insertions containing long complements to splice sites and a branch point inhibited splicing in vitro but not in vivo. These results raise the possibility that in cells most pre-mRNA secondary structures either are not maintained long enough to influence splicing choices, or never form at all.

scholarly journals Dynamic changes in RNA–protein interactions and RNA secondary structure in mammalian erythropoiesis

2021 ◽  
Vol 4 (9) ◽  
pp. e202000659
Author(s):  
Mengge Shan ◽  
Xinjun Ji ◽  
Kevin Janssen ◽  
Ian M Silverman ◽  
Jesse Humenik ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Protein Interactions ◽  
Rna Secondary Structure ◽  
Rna Binding ◽  
Developmental Process ◽  
Global Analysis ◽  
Sequence Motifs ◽  
Rna Molecules ◽  
Interaction Sites ◽  
Rna Interaction

Two features of eukaryotic RNA molecules that regulate their post-transcriptional fates are RNA secondary structure and RNA-binding protein (RBP) interaction sites. However, a comprehensive global overview of the dynamic nature of these sequence features during erythropoiesis has never been obtained. Here, we use our ribonuclease-mediated structure and RBP-binding site mapping approach to reveal the global landscape of RNA secondary structure and RBP–RNA interaction sites and the dynamics of these features during this important developmental process. We identify dynamic patterns of RNA secondary structure and RBP binding throughout the process and determine a set of corresponding protein-bound sequence motifs along with their dynamic structural and RBP-binding contexts. Finally, using these dynamically bound sequences, we identify a number of RBPs that have known and putative key functions in post-transcriptional regulation during mammalian erythropoiesis. In total, this global analysis reveals new post-transcriptional regulators of mammalian blood cell development.

scholarly journals Analysis of the Secondary Structure of β-Amyloid (Aβ42) Fibrils by Systematic Proline Replacement

2004 ◽  
Vol 279 (50) ◽  
pp. 52781-52788 ◽  
Author(s):  
Akira Morimoto ◽  
Kazuhiro Irie ◽  
Kazuma Murakami ◽  
Yuichi Masuda ◽  
Hajime Ohigashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Amyloid Fibrils ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Aβ Peptides ◽  
Amyloid Peptides ◽  
Aggregation Inhibitors ◽  
Β Amyloid ◽  
Β Sheet

Amyloid fibrils in Alzheimer's disease mainly consist of 40- and 42-mer β-amyloid peptides (Aβ40 and Aβ42) that exhibit aggregative ability and neurotoxicity. Although the aggregates of Aβ peptides are rich in intermolecular β-sheet, the precise secondary structure of Aβ in the aggregates remains unclear. To identify the amino acid residues involved in the β-sheet formation, 34 proline-substituted mutants of Aβ42 were synthesized and their aggregative ability and neurotoxicity on PC12 cells were examined. Prolines are rarely present in β-sheet, whereas they are easily accommodated in β-turn as a Pro-Xcorner. Among the mutants at positions 15-32, only E22P-Aβ42 extensively aggregated with stronger neurotoxicity than wild-type Aβ42, suggesting that the residues at positions 15-21 and 24-32 are involved in the β-sheet and that the turn at positions 22 and 23 plays a crucial role in the aggregation and neurotoxicity of Aβ42. The C-terminal proline mutants (A42P-, I41P-, and V40P-Aβ42) hardly aggregated with extremely weak cytotoxicity, whereas the C-terminal threonine mutants (A42T- and I41T-Aβ42) aggregated potently with significant cytotoxicity. These results indicate that the hydrophobicity of the C-terminal two residues of Aβ42 is not related to its aggregative ability and neurotoxicity, rather the C-terminal three residues adopt the β-sheet. These results demonstrate well the large difference in aggregative ability and neurotoxicity between Aβ42 and Aβ40. In contrast, the proline mutants at the N-terminal 13 residues showed potent aggregative ability and neurotoxicity similar to those of wild-type Aβ42. The identification of the β-sheet region of Aβ42 is a basis for designing new aggregation inhibitors of Aβ peptides.

In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features

Nature ◽  
2013 ◽  
Vol 505 (7485) ◽  
pp. 696-700 ◽  
Author(s):  
Yiliang Ding ◽  
Yin Tang ◽  
Chun Kit Kwok ◽  
Yu Zhang ◽  
Philip C. Bevilacqua ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Rna Secondary Structure ◽  
Genome Wide

scholarly journals 3′UTR SL-IV and DB1 Regions Contribute to Japanese Encephalitis Virus Replication and Pathogenicity

2021 ◽  
Vol 8 ◽  
Author(s):  
Jinchao Xing ◽  
Youyue Zhang ◽  
Ziying Lin ◽  
Lele Liu ◽  
Qiang Xu ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Rna Secondary Structure ◽  
Rational Design ◽  
Case Fatality ◽  
Encephalitis Virus ◽  
Reverse Genetic System ◽  
Emerging Pathogens

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV is maintained in an enzootic cycle and causes reproductive failure in pigs. Notably, the shift in JEV genotypes is not fully protected by existing vaccines, so the development of a candidate vaccine is urgently needed. In this study, we compared pathogenicity between Japanese encephalitis virus SA14 and BJB (isolated from humans in the 1970s) strains. We found that the BJB strain was attenuated in mice and that there was no case fatality rate. The growth rate of BJB was higher than SA14 virus in BHK-21 cells. Based on the sequence alignment of the viral genome between the SA14 and BJB virus strains, some mutations at sites 248, 254, 258, and 307 were observed in the 3′ untranslated region (3′UTR). The 3′UTR of JEV plays a very important role in the viral life cycle. Furthermore, using a reverse genetic system, we conducted and rescued the parental JEV strain SA14 (T248, A254, and A258) and the mutant virus rSA14-3′UTRmut (T248C, A254G, A258G, and 307G). Through an analysis of the RNA secondary structure model of the 3′UTR, we discovered that the mutations of T248C, A254G, and A258G reduced the apiculus ring and increased the lateral ring significantly in the stem-loop structures IV (SL-IV) structure region of 3′UTR. Moreover, the insertion of 307G added a ring to the dumbbell structure 1 (DB1) structure region. Strikingly, these RNA secondary structure changes in 3′UTR of rSA14-3′UTRmut increased viral negative chain RNA production and enhanced the replication ability of the virus in BHK-21 cells. However, in vivo mouse experiments illustrated that the rSA14-3′UTRmut virus significantly decreased the neurovirulence of JEV. These results affirmed that the JEV SL-IV and DB1 regions play an important role in viral proliferation and pathogenicity. Taken together, we complement the study of RNA element function in the 3′UTR region of JEV by providing a new target for the rational design of live attenuated candidate vaccines and the increase of virus production.

scholarly journals Essential Role of Cyclization Sequences in Flavivirus RNA Replication

2001 ◽  
Vol 75 (14) ◽  
pp. 6719-6728 ◽  
Author(s):  
Alexander A. Khromykh ◽  
Hedije Meka ◽  
Kimberley J. Guyatt ◽  
Edwin G. Westaway
Keyword(s):  
Secondary Structure ◽  
Rna Secondary Structure ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Genomic Region ◽  
Virus Species ◽  
Base Pairing ◽  
Complementary Sequences ◽  
Covalently Linked

ABSTRACT A possible role in RNA replication for interactions between conserved complementary (cyclization) sequences in the 5′- and 3′-terminal regions of Flavivirus RNA was previously suggested but never tested in vivo. Using the M-fold program for RNA secondary-structure predictions, we examined for the first time the base-pairing interactions between the covalently linked 5′ genomic region (first ∼160 nucleotides) and the 3′ untranslated region (last ∼115 nucleotides) for a range of mosquito-borneFlavivirus species. Base-pairing occurred as predicted for the previously proposed conserved cyclization sequences. In order to obtain experimental evidence of the predicted interactions, the putative cyclization sequences (5′ or 3′) in the replicon RNA of the mosquito-borne Kunjin virus were mutated either separately, to destroy base-pairing, or simultaneously, to restore the complementarity. None of the RNAs with separate mutations in only the 5′ or only the 3′ cyclization sequences was able to replicate after transfection into BHK cells, while replicon RNA with simultaneous compensatory mutations in both cyclization sequences was replication competent. This was detected by immunofluorescence for expression of the major nonstructural protein NS3 and by Northern blot analysis for amplification and accumulation of replicon RNA. We then used the M-fold program to analyze RNA secondary structure of the covalently linked 5′- and 3′-terminal regions of three tick-borne virus species and identified a previously undescribed additional pair of conserved complementary sequences in locations similar to those of the mosquito-borne species. They base-paired with ΔG values of approximately −20 kcal, equivalent or greater in stability than those calculated for the originally proposed cyclization sequences. The results show that the base-pairing between 5′ and 3′ complementary sequences, rather than the nucleotide sequence per se, is essential for the replication of mosquito-borne Kunjin virus RNA and that more than one pair of cyclization sequences might be involved in the replication of the tick-borne Flavivirusspecies.

scholarly journals Predicting in vivo binding sites of RNA-binding proteins using mRNA secondary structure

RNA ◽  
2010 ◽  
Vol 16 (6) ◽  
pp. 1096-1107 ◽  
Author(s):  
X. Li ◽  
G. Quon ◽  
H. D. Lipshitz ◽  
Q. Morris
Keyword(s):  
Secondary Structure ◽  
Binding Sites ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Secondary Structure ◽  
In Vivo Binding
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