R-loop Research Articles

Preferential formation of Z-RNA over intercalated motifs in long noncoding RNA.

Secondary structure is a principal determinant of lncRNA function, predominantly regarding scaffold formation and interfaces with target molecules. Noncanonical secondary structures that form in nucleic acids have known roles in regulating gene expression and include G-quadruplexes (G4s), intercalated motifs (iMs), and R-loops (RLs). In this paper, we used the computational tools G4-iM Grinder and QmRLFS-finder to predict the formation of each of these structures throughout the lncRNA transcriptome in comparison to protein-coding transcripts. The importance of the predicted structures in lncRNAs in biological contexts was assessed by combining our results with publicly available lncRNA tissue expression data followed by pathway analysis. The formation of predicted G4 (pG4) and iM (piM) structures in select lncRNA sequences was confirmed in vitro using biophysical experiments under near-physiological conditions. We find that the majority of the tested pG4s form highly stable G4 structures, and identify many previously unreported G4s in biologically important lncRNAs. In contrast, none of the piM sequences are able to form iM structures, consistent with the idea that RNA is unable to form stable iMs. Unexpectedly, these C-rich sequences instead form Z-RNA structures, which have not been previously observed in regions containing cytosine repeats and represent an interesting and underexplored target for protein-RNA interactions. Our results highlight the prevalence and potential structure-associated functions of noncanonical secondary structures in lncRNAs, and show G4 and Z-RNA structure formation in many lncRNA sequences for the first time, furthering the understanding of the structure-function relationship in lncRNAs.

SWI/SNF complexes as determinants of R-loop metabolism.

SWI/SNF complexes as determinants of R-loop metabolism.

DNA:RNA hybrids at telomeres - when it is better to be out of the (R) loop.

R-loops (RLs) are three-stranded nucleic acid structures that contain a DNA:RNA hybrid and a displaced DNA strand. Genomic regions with GC skew and a G-rich transcript are particularly prone to form RLs. RLs play important physiological roles in cells; however, when present at abnormally high levels, they may threaten genome stability. The perfect GC skew of telomeric repeats and the discovery of telomeric repeat-containing RNA (TERRA), a long noncoding transcript that consists of the G-rich telomeric sequence, make telomeric sequences the perfect candidates for generating RLs. Indeed, in the past 5years, telomere R-loops (TRLs) have been demonstrated in Saccharomycescerevisiae, Trypanosomabrucei, and human cells. The presence of TRLs in normal human cells that transcribe low levels of TERRA, suggests a physiological role for these nucleic structures in telomere maintenance. Abnormally enhanced TERRA transcription, as found in several human pathological conditions, leads to high TRL levels and various cellular outcomes, depending on the recombinogenic capabilities of the cells. Study of TRLs in various organisms highlights the necessity for tight regulation of these structures, which can switch from beneficial to detrimental under different conditions. Here, we review the current state of knowledge on TRLs, describe several means by which TRLs are regulated, and discuss how findings from yeast are relevant to human pathological scenarios in which TRLs are deregulated.

Transcription: Putting R loops firmly on the map.

Transcription: Putting R loops firmly on the map.

Structural basis for guide RNA processing and seed-dependent DNA targeting and cleavage by CRISPR-Cas12a

The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs, and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a in a binary complex with a guide RNA, and in a R-loop complex containing a non-cleavable guide RNA precursor and full-length target DNA. Corroborated by biochemical experiments, these structures elucidate the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. The R-loop complex structure furthermore reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes that may contribute to further development of genome editing technologies.

R-loop structure: the formation and the effects on genomic stability.

R-loop is a type of three-stranded nucleic acid structure that is made up of an RNA:DNA hybrid, formed due to failing separation of a nascent RNA molecule with transcripting template in transcription or by the re-annealing of RNA molecule with one of the two strands in a double stranded DNA molecule, along with the single stranded DNA, which is either the non-template strand in the transcription bubble or the RNA substituted DNA strand. Formation of R-loops can occur when transcription goes through a genomic DNA region having a tract of G bases in the non-template strand in the transcription bubble or through a type of triplet microsatellite DNA sequences that are known to be associated with certain human diseases. The negative supercoiling forces accumulated in the transcription bubble, and the misprocessing of RNA precursors, as well as the delayed utilizations and transportations of RNA molecules to cytoplasm promote R loop formation. Many studies show that cells can manage R loop formation with efficiency, and can also process the R-loops already formed in the cell, and by which, the bad effects of R-loops on DNA replication, gene mutation and homologous recombination can be regulated. In this review, we summarize the formation and the impacts of R-loops on DNA replication, mutation rates and the frequencies of homologous recombination, and also discusse the possible role of the R-loop induced DNA replication in mediating trinucleotide repeat expansions as seen with those frequently associated with human neuromuscular degenerative diseases.

Processed switch-region transcripts and retrotranscripts as possible generators of heteroduplex 'R-loop' targets for AID deamination

Processed switch-region transcripts and retrotranscripts as possible generators of heteroduplex 'R-loop' targets for AID deamination

Transcription-dependent R-loop formation at mammalian class switch sequences.

Transcription-dependent R-loop formation at mammalian class switch sequences.

3-D experimental identification of force systems from orthodontic loops activated for first order corrections.

Intra-arch irregularities can be corrected using wire of low stiffness, wires of increasing stiffnesses, or by the activation of loops built into the appliance. While the orthodontist controls only the magnitude of force when leveling with continuous arches, the configuration and positioning of loops offer the possibility of controlling the type and direction of force. In the present study, force systems developed by the L-loop, the T-loop, and the rectangular (R-) loop were analyzed with respect to the force systems acting for first order irregularities, buccolingual movement, and rotation along the long axis of the tooth. An interbracket distance of 21 mm was chosen, and the loops were analyzed in a testing machine that made it possible to register forces and moments simultaneously in three planes of space. The activations included a symmetrical translation of 1 mm made in steps of .2 mm, corresponding to a buccolingual movement, and 10-degree rotations clockwise and counterclockwise in steps of one degree. Force systems were recorded during activation and deactivation. Loops made of TMA wire delivered 40% of the force delivered by the same loops made of stainless steel wire. The T-loop generated a force system that deviated qualitatively only slightly from that delivered by a straight wire. The L-loop generated a force system that was dependent on orientation; constancy was better corresponding to the anterior part of the loop. It was evident that the rectangular loop was capable of generating any desired moment-to-force ratio, and the R-loop demonstrated a high degree of constancy of the force system. Rectangular loops should, therefore, be preferred for making first order corrections.