scholarly journals How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing

2017 ◽  
Vol 63 (4) ◽  
Author(s):  
Natalia Koralewska ◽  
Weronika Hoffmann ◽  
Maria Pokornowska ◽  
Marek Cezary Milewski ◽  
Andrea Lipinska ◽  
...  
Keyword(s):  
Rna Binding ◽  
Feedback Loops ◽  
Small Interfering Rnas ◽  
Binding Studies ◽  
Hairpin Rna ◽  
Rna Molecules ◽  
Short Rnas ◽  
Microrna Processing

Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by variety of proteins are being elucidated, less is known about non-protein factors; e.g. RNA molecules, that may influence the enzyme activity. Therefore, we decided to investigate the problem of whether the RNA molecules can function not only as Dicer substrates but also as its regulators.Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding with the enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.

scholarly journals Trafficking of siRNA precursors by the dsRBD protein Blanks in Drosophila

2020 ◽  
Vol 48 (7) ◽  
pp. 3906-3921 ◽  
Author(s):  
Volker Nitschko ◽  
Stefan Kunzelmann ◽  
Thomas Fröhlich ◽  
Georg J Arnold ◽  
Klaus Förstemann
Keyword(s):  
Small Rnas ◽  
Rna Binding ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Binding Domains ◽  
Convergent Transcription ◽  
Dsrna Binding ◽  
Functional Screens

Abstract RNA interference targets aberrant transcripts with cognate small interfering RNAs, which derive from double-stranded RNA precursors. Several functional screens have identified Drosophila blanks/lump (CG10630) as a facilitator of RNAi, yet its molecular function has remained unknown. The protein carries two dsRNA binding domains (dsRBD) and blanks mutant males have a spermatogenesis defect. We demonstrate that blanks selectively boosts RNAi triggered by dsRNA of nuclear origin. Blanks binds dsRNA via its second dsRBD in vitro, shuttles between nucleus and cytoplasm and the abundance of siRNAs arising at many sites of convergent transcription is reduced in blanks mutants. Since features of nascent RNAs - such as introns and transcription beyond the polyA site – contribute to the small RNA pool, we propose that Blanks binds dsRNA formed by cognate nascent RNAs in the nucleus and fosters its export to the cytoplasm for dicing. We refer to the resulting small RNAs as blanks exported siRNAs (bepsiRNAs). While bepsiRNAs were fully dependent on RNA binding to the second dsRBD of blanks in transgenic flies, male fertility was not. This is consistent with a previous report that linked fertility to the first dsRBD of Blanks. The role of blanks in spermatogenesis appears thus unrelated to its role in dsRNA export.

scholarly journals Oncogenic lncRNA ZNF561-AS1 is essential for colorectal cancer proliferation and survival through regulation of miR-26a-3p/miR-128-5p-SRSF6 axis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Zizhen Si ◽  
Lei Yu ◽  
Haoyu Jing ◽  
Lun Wu ◽  
Xidi Wang
Keyword(s):  
Colorectal Cancer ◽  
Rna Binding ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Cancer Proliferation ◽  
Mouse Xenograft ◽  
Mouse Xenograft Model

Abstract Background Long non-coding RNAs (lncRNA) are reported to influence colorectal cancer (CRC) progression. Currently, the functions of the lncRNA ZNF561 antisense RNA 1 (ZNF561-AS1) in CRC are unknown. Methods ZNF561-AS1 and SRSF6 expression in CRC patient samples and CRC cell lines was evaluated through TCGA database analysis, western blot along with real-time PCR. SRSF6 expression in CRC cells was also examined upon ZNF561-AS1 depletion or overexpression. Interaction between miR-26a-3p, miR-128-5p, ZNF561-AS1, and SRSF6 was examined by dual luciferase reporter assay, as well as RNA binding protein immunoprecipitation (RIP) assay. Small interfering RNA (siRNA) mediated knockdown experiments were performed to assess the role of ZNF561-AS1 and SRSF6 in the proliferative actives and apoptosis rate of CRC cells. A mouse xenograft model was employed to assess tumor growth upon ZNF561-AS1 knockdown and SRSF6 rescue. Results We find that ZNF561-AS1 and SRSF6 were upregulated in CRC patient tissues. ZNF561-AS1 expression was reduced in tissues from treated CRC patients but upregulated in CRC tissues from relapsed patients. SRSF6 expression was suppressed and enhanced by ZNF561-AS1 depletion and overexpression, respectively. Mechanistically, ZNF561-AS1 regulated SRSF6 expression by sponging miR-26a-3p and miR-128-5p. ZNF561-AS1-miR-26a-3p/miR-128-5p-SRSF6 axis was required for CRC proliferation and survival. ZNF561-AS1 knockdown suppressed CRC cell proliferation and triggered apoptosis. ZNF561-AS1 depletion suppressed the growth of tumors in a model of a nude mouse xenograft. Similar observations were made upon SRSF6 depletion. SRSF6 overexpression reversed the inhibitory activities of ZNF561-AS1 in vivo, as well as in vitro. Conclusion In summary, we find that ZNF561-AS1 promotes CRC progression via the miR-26a-3p/miR-128-5p-SRSF6 axis. This study reveals new perspectives into the role of ZNF561-AS1 in CRC.

scholarly journals RNA Helicase A Regulates the Replication of RNA Viruses

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 361
Author(s):  
Rui-Zhu Shi ◽  
Yuan-Qing Pan ◽  
Li Xing
Keyword(s):  
Virus Replication ◽  
Rna Binding ◽  
Rna Viruses ◽  
Rna Helicase ◽  
Rna Helicase A ◽  
Double Stranded Rna ◽  
Binding Domains ◽  
N Terminus

The RNA helicase A (RHA) is a member of DExH-box helicases and characterized by two double-stranded RNA binding domains at the N-terminus. RHA unwinds double-stranded RNA in vitro and is involved in RNA metabolisms in the cell. RHA is also hijacked by a variety of RNA viruses to facilitate virus replication. Herein, this review will provide an overview of the role of RHA in the replication of RNA viruses.

scholarly journals The Regulator of Nitrate Assimilation in Ascomycetes Is a Dimer Which Binds a Nonrepeated, Asymmetrical Sequence

1998 ◽  
Vol 18 (3) ◽  
pp. 1339-1348 ◽  
Author(s):  
Joseph Strauss ◽  
M. Isabel Muro-Pastor ◽  
Claudio Scazzocchio
Keyword(s):  
Nitrate Assimilation ◽  
Binding Studies ◽  
Binding Ability ◽  
Zinc Cluster ◽  
First Case ◽  
Vitro Binding ◽  
Nitrate Induction ◽  
Dna Complex

ABSTRACT The regulation of nitrate assimilation seems to follow the same pattern in all ascomycetes where this process has been studied. We show here by in vitro binding studies and a number of protection and interference techniques that the transcription factor mediating nitrate induction in Aspergillus nidulans, a protein containing a binuclear zinc cluster DNA binding domain, recognizes an asymmetrical sequence of the form CTCCGHGG. We further show that the protein binds to its consensus site as a dimer. We establish the role of the putative dimerization element by its ability to replace the analogous element of the cI protein of phage λ. Mutagenesis of crucial leucines of the dimerization element affect both the binding ability of the dimer and the conformation of the resulting protein-DNA complex. This is the first case to be described where a dimer recognizes such an asymmetrical nonrepeated sequence, presumably by each monomeric subunit making different contacts with different DNA half-sites.

scholarly journals The infinite possibilities of RNA therapeutics

2021 ◽  
Author(s):  
Evelyn C Mollocana-Lara ◽  
Ming Ni ◽  
Spiros N Agathos ◽  
Fernando A Gonzales-Zubiate
Keyword(s):  
Literature Review ◽  
Antisense Oligonucleotides ◽  
State Of The Art ◽  
Small Interfering Rnas ◽  
New Era ◽  
Final Point ◽  
Clinical Investigations ◽  
General Aspects

Abstract Although the study of ribonucleic acid (RNA) therapeutics started decades ago, for many years, this field of research was overshadowed by the growing interest in DNA-based therapies. Nowadays, the role of several types of RNA in cell regulation processes and the development of various diseases have been elucidated, and research in RNA therapeutics is back with force. This short literature review aims to present general aspects of many of the molecules currently used in RNA therapeutics, including in vitro transcribed mRNA (IVT mRNA), antisense oligonucleotides (ASOs), aptamers, small interfering RNAs (siRNAs), and microRNAs (miRNAs). In addition, we describe the state of the art of technologies applied for synthetic RNA manufacture and delivery. Likewise, we detail the RNA-based therapies approved by the FDA so far, as well as the ongoing clinical investigations. As a final point, we highlight the current and potential advantages of working on RNA-based therapeutics and how these could lead to a new era of accessible and personalized healthcare.

scholarly journals RNA binding to human METTL3-METTL14 restricts N6-deoxyadenosine methylation of DNA in vitro

2022 ◽  
Author(s):  
Shan Qi ◽  
Javier Mota ◽  
Siu-Hong Chan ◽  
Johanna Villarreal ◽  
Nan Dai ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Rna Binding ◽  
Methyl Group ◽  
High Affinity ◽  
Methylation Of Dna ◽  
Dna And Rna ◽  
Close Proximity ◽  
Methylation Activity

Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from S-adenosyl-L-methionine to N6 amino group of adenosine bases in RNA (m6A) and DNA (m6dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m6dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m6A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely restricted by structured RNA elements prevalent in long noncoding and other cellular RNAs.

scholarly journals The Emerging Role and Promise of Circular RNAs in Obesity and Related Metabolic Disorders

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1473
Author(s):  
Mohamed Zaiou
Keyword(s):  
Rna Binding ◽  
Metabolic Diseases ◽  
Rna Binding Proteins ◽  
In Vivo Studies ◽  
Future Research ◽  
Circular Rnas ◽  
Exciting Field ◽  
Rna Molecules

Circular RNAs (circRNAs) are genome transcripts that are produced from back-splicing of specific regions of pre-mRNA. These single-stranded RNA molecules are widely expressed across diverse phyla and many of them are stable and evolutionary conserved between species. Growing evidence suggests that many circRNAs function as master regulators of gene expression by influencing both transcription and translation processes. Mechanistically, circRNAs are predicted to act as endogenous microRNA (miRNA) sponges, interact with functional RNA-binding proteins (RBPs), and associate with elements of the transcriptional machinery in the nucleus. Evidence is mounting that dysregulation of circRNAs is closely related to the occurrence of a range of diseases including cancer and metabolic diseases. Indeed, there are several reports implicating circRNAs in cardiovascular diseases (CVD), diabetes, hypertension, and atherosclerosis. However, there is very little research addressing the potential role of these RNA transcripts in the occurrence and development of obesity. Emerging data from in vitro and in vivo studies suggest that circRNAs are novel players in adipogenesis, white adipose browning, obesity, obesity-induced inflammation, and insulin resistance. This study explores the current state of knowledge on circRNAs regulating molecular processes associated with adipogenesis and obesity, highlights some of the challenges encountered while studying circRNAs and suggests some perspectives for future research directions in this exciting field of study.

scholarly journals Sequence diversity in the 3’ untranslated region of alphavirus modulates IFIT2-dependent restriction in a cell type-dependent manner

2021 ◽  
Author(s):  
Sarah E. Hickson ◽  
Eden Brekke ◽  
Johannes Schwerk ◽  
Indraneel Saluhke ◽  
Shivam Zaver ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Rna Binding ◽  
Structural Features ◽  
Differential Sensitivity ◽  
Restriction Factor ◽  
Dependent Manner ◽  
Tetratricopeptide Repeats ◽  
Rna Interaction

ABSTRACTAlphaviruses (family Togaviridae) are a diverse group of positive-sense RNA (+ssRNA) viruses that are transmitted by arthropods and are the causative agent of several significant human and veterinary diseases. Interferon (IFN)-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding IFN stimulated genes (ISGs) that are highly upregulated following viral infection, and have been identified as potential restrictors of alphaviruses. The mechanism by which IFIT1 restricts RNA viruses is dependent on self and non-self-discrimination of RNA, and alphaviruses evade this recognition via their 5’UTR. However, the role of IFIT2 during alphavirus replication and the mechanism of viral replication inhibition is unclear. In this study, we identify IFIT2 as a restriction factor for Venezuelan equine encephalitis virus (VEEV) and show that IFIT2 binds the 3’ untranslated region (3’UTR) of the virus. We investigated the potential role of variability in the 3’UTR of the virus affecting IFIT2 antiviral activity by studying infection with VEEV. Comparison of recombinant VEEV clones containing 3’UTR sequences derived from epizootic and enzootic isolates exhibited differential sensitivity to IFIT2 restriction in vitro infection studies, suggesting that the alphavirus 3’UTR sequence may function in part to evade IFIT2 restriction. In vitro binding assays demonstrate that IFIT2 binds to the VEEV 3’UTR, however in contrast to previous studies VEEV restriction did not appear to be dependent on the ability of IFIT2 to inhibit translation of viral RNA, suggesting a novel mechanism of IFIT2 restriction. Our study demonstrates that IFIT2 is a restriction factor for alphaviruses and variability in the 3’UTR of VEEV can modulate viral restriction by IFIT2. Ongoing studies are exploring the biological consequences of IFIT2-VEEV RNA interaction in viral pathogenesis and defining sequence and structural features of RNAs that regulate IFIT2 recognition.

scholarly journals iCLIP analysis of RNA substrates of the archaeal exosome

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Jochen Bathke ◽  
A. Susann Gauernack ◽  
Oliver Rupp ◽  
Lennart Weber ◽  
Christian Preusser ◽  
...  
Keyword(s):  
Rna Binding ◽  
Transcript Abundance ◽  
Global Scale ◽  
Circular Rnas ◽  
Rna Seq ◽  
Uv Crosslinking ◽  
Rna Targets ◽  
Nucleotide Resolution

Abstract Background The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3′ to 5′ direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3′-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging. Results To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon Sulfolobus solfataricus. A relatively high proportion (17–19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5′ parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3′-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5′-ends of RNAs was detected. Conclusions In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5′-ends of genes suggests simultaneous binding of both RNA ends by the S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3′-5′ direction.

Alternate promoters and variable splicing lead to hNedd4–2 isoforms with a C2 domain and varying number of WW domains

2003 ◽  
Vol 285 (5) ◽  
pp. F916-F929 ◽  
Author(s):  
Omar A. Itani ◽  
Jason R. Campbell ◽  
Juan Herrero ◽  
Peter M. Snyder ◽  
Christie P. Thomas
Keyword(s):  
Collecting Duct ◽  
Rnase Protection Assay ◽  
C2 Domain ◽  
Binding Studies ◽  
Specific Expression ◽  
Rnase Protection ◽  
Ww Domains ◽  
Rat Thyroid

Mutations that disrupt a PY motif in epithelial Na+ channel (ENaC) subunits increase surface expression of Na+ channels in the collecting duct, resulting in greater Na+ reabsorption. Recently, Nedd4 and Nedd4–2 have been identified as ubiquitin ligases that can interact with ENaC via its PY motifs to regulate channel activity. To further understand the role of human Nedd4–2 (hNedd4–2), we cloned its cDNAs and determined its genomic organization using a bioinformatic approach. The gene is present as a single copy, spans at least 400 kb, and contains >40 exons. Multiple 5′-exons were identified by 5′-rapid amplification of cDNA ends, and tissue-specific expression of these transcripts was noted by RT-PCR and RNase protection assay. Alternate polyadenylation signal sequences led to varying lengths of the 3′-untranslated region. Alternate splicing events within internal exons were also noted. Open reading frame analysis indicates that hNedd4–2 encode multiple protein variants with and without a C2 domain, and with a variable number of WW domains. Coexpression, in Fischer rat thyroid epithelia, of ENaC and Nedd4–2 cDNAs leads to a significant reduction in amiloride-sensitive currents, confirming a role in Na+ transport regulation. In vitro binding studies demonstrated that individual PY motifs of α-, β-, and γ-ENaC have strong affinity for WW domains 3 and 4 but not 1 and 2. These studies indicate that alternate transcripts of Nedd4–2 may interact with ENaC differently. Understanding the function of variant proteins will increase our knowledge of the role of hNedd4–2 in the regulation of ENaC and define protein domains important for Nedd4–2 function.

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