Correction to: A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR.

In Volume 46 of the Journal of Biosciences, in the article titled 'A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR' by Ridim D Mote, V Shinde Laxmikant, Surya Bansi Singh, Mahak Tiwari, Hemant Singh, Juhi Srivastava, Vidisha Tripathi,Vasudevan Seshadri, Amitabha Majumdar and Deepa Subramanyam, published on 27 November 2021 (https://doi.org/10.1007/s12038-021- 00231-w), the second author's name was incorrectly set as V Shinde Laxmikant. The correct name should read as Shinde Laxmikant V.

A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR.

Real-time PCR is a widely used technique for quantification of gene expression. However, commercially available kits for real-time PCR are very expensive. The ongoing coronavirus pandemic has severely hampered the economy in a number of developing countries, resulting in a reduction in available research funding. The fallout of this will result in limiting educational institutes and small enterprises from using cutting edge biological techniques such as real-time PCR. Here, we report a cost-effective approach for preparing and assembling cDNA synthesis and real-time PCR mastermixes with similar efficiencies as commercially available kits. Our results thus demonstrate an alternative to commercially available kits.

The S-phase-induced lncRNA SUNO1 promotes cell proliferation by controlling YAP1/Hippo signaling pathway.

Cell cycle is a cellular process that is subject to stringent control. In contrast to the wealth of knowledge of proteins controlling the cell cycle, very little is known about the molecular role of lncRNAs (long noncoding RNAs) in cell-cycle progression. By performing genome-wide transcriptome analyses in cell-cycle-synchronized cells, we observed cell-cycle phase-specific induction of >2000 lncRNAs. Further, we demonstrate that an S-phase-upregulated lncRNA, SUNO1, facilitates cell-cycle progression by promoting YAP1-mediated gene expression. SUNO1 facilitates the cell-cycle-specific transcription of WTIP, a positive regulator of YAP1, by promoting the co-activator, DDX5-mediated stabilization of RNA polymerase II on chromatin. Finally, elevated SUNO1 levels are associated with poor cancer prognosis and tumorigenicity, implying its pro-survival role. Thus, we demonstrate the role of a S-phase up-regulated lncRNA in cell-cycle progression via modulating the expression of genes controlling cell proliferation.

Antagonism between splicing and microprocessor complex dictates the serum-induced processing of lnc-MIRHG for efficient cell cycle reentry.

Cellular quiescence and cell cycle reentry regulate vital biological processes such as cellular development and tissue homeostasis and are controlled by precise regulation of gene expression. The roles of long noncoding RNAs (lncRNAs) during these processes remain to be elucidated. By performing genome-wide transcriptome analyses, we identify differential expression of several hundreds of lncRNAs, including a significant number of the less-characterized class of microRNA-host-gene (MIRHG) lncRNAs or lnc-MIRHGs, during cellular quiescence and cell cycle reentry in human diploid fibroblasts. We observe that MIR222HG lncRNA displays serum-stimulated RNA processing due to enhanced splicing of the host nascent pri-MIR222HG transcript. The pre-mRNA splicing factor SRSF1 negatively regulates the microprocessor-catalyzed cleavage of pri-miR-222, thereby increasing the cellular pool of the mature MIR222HG Association of SRSF1 to pri-MIR222HG, including to a mini-exon, which partially overlaps with the primary miR-222 precursor, promotes serum-stimulated splicing over microRNA processing of MIR222HG Further, we observe that the increased levels of spliced MIR222HG in serum-stimulated cells promote the cell cycle reentry post quiescence in a microRNA-independent manner. MIR222HG interacts with DNM3OS, another lncRNA whose expression is elevated upon serum-stimulation, and promotes cell cycle reentry. The double-stranded RNA binding protein ILF3/2 complex facilitates MIR222HG:DNM3OS RNP complex assembly, thereby promoting DNM3OS RNA stability. Our study identifies a novel mechanism whereby competition between the splicing and microprocessor machinery modulates the serum-induced RNA processing of MIR222HG, which dictates cell cycle reentry.

Enhancement of hydrophilicity, biocompatibility and biodegradability of poly(ε-caprolactone) electrospun nanofiber scaffolds using poly(ethylene glycol) and poly(L-lactide-co-ε-caprolactone-co-glycolide) as additives for soft tissue engineering.

In this study, poly(ε-caprolactone) (PCL) has been blended with a more hydrophilic poly(ethylene glycol) (PEG) and with a biocompatible block-co-polymer: poly(L-lactide-co-ε-caprolactone-co-glycolide) (PLCG) in order to improve hydrophilicity, biocompatibility and biodegradability of PCL. PCL and the blend solutions were subjected to electrospinning to produce nanofiber scaffolds by the addition of only 1 wt% of PEG and PLCG either singly or in combination in PCL to retain the mechanical properties of the scaffolds. PCL-PEG-PLCG ternary and two binary (PCL-PEG and PCL-PLCG) blend nanofiber scaffolds have been prepared for comparison. The resulting nanofibers showed a smooth and flaw-free surface and the diameter of the nanofibers displayed a normal distribution. The PCL-PEG nanofiber scaffold showed improved hydrophilicity [water contact angle (WCA) ∼84°] over pristine PCL (WCA ∼127°); while PCL-PLCG and PCL-PEG-PLCG scaffolds exhibited absolute wetting by water, likely due to high porosity. In vitro biocompatibility studies using gingival mesenchymal stem cells (gMSCs) suggested that, both the PCL and the blend scaffolds were biocompatible supporting cell-viability and growth of gMSCs following their seeding on these scaffolds. Biodegradation studies in phosphate buffer solution showed that the addition of PEG and PLCG in PCL increased the weight loss of scaffolds with time, indicating higher extent of biodegradation in the blend scaffolds and the weight loss followed the power law curve with time.

Conservation of Ovary-Specific Genes, Foxl2, Aromatase, and Rspo1, in the Common Indian Garden Lizard, Calotes versicolor, That Lacks Chromosomal or Temperature-Dependent Sex Determination.

Foxl2,Rspo1, and Aromatase are genes important in the ovary developmental pathway in mammals and birds. Here, we show their presence in the lizard, Calotes versicolor, which is known to lack a chromosomal as well as a temperature-dependent mode of sex determination and has an indeterminate, bipotential gonad throughout embryonic development. The expression of the 3 genes, as well as that of CvSox9 and Wnt4 - the known testis and ovary pathway genes - was studied by RT-PCR and whole tissue RNA in situ hybridization (WRISH) on the developing mesonephros gonadal complex (MGC). The expression of all 3 genes was initiated in the gonad shortly after its evagination from the mesonephros (day 5 onwards). CvFoxl2 generally was expressed in those MGCs in which CvSox9 was either not expressed or lowly expressed and vice versa. On the other hand, CvArom was expressed rather sporadically and randomly, showing no association with CvFoxl2, CvRspo1, or CvSox9, though in later stages WRISH preparations showed its coincidence with CvWnt4. CvRspo1 was expressed in almost all embryos right from day 5. Immunofluorescence localization of Rspo1 and Foxl2 proteins showed their presence in the gonads from day 10 onwards, and by day 25 it was primarily confined to the cortex but away from the coelomic epithelium of the gonadal cortex. Apparently both proteins were localized in the pregranulosa cells, Rspo1 in the cytoplasm and Foxl2 in the nucleus. Thus, it is clear that both CvFoxl2 and CvRspo1 are active in ovary formation, but whether they are expressed in the same or different cells is unknown. Though the transcription pattern of CvArom remains circumspect for its role in differentiation of the ovary, earlier evidence on aromatase inhibitor-induced reversal to the male sex indicates its importance in ovary function.

MIR100 host gene-encoded lncRNAs regulate cell cycle by modulating the interaction between HuR and its target mRNAs.

Long non-coding RNAs (lncRNAs) regulate vital biological processes, including cell proliferation, differentiation and development. A subclass of lncRNAs is synthesized from microRNA (miRNA) host genes (MIRHGs) due to pre-miRNA processing, and are categorized as miRNA-host gene lncRNAs (lnc-miRHGs). Presently, the cellular function of most lnc-miRHGs is not well understood. We demonstrate a miRNA-independent role for a nuclear-enriched lnc-miRHG in cell cycle progression. MIR100HG produces spliced and stable lncRNAs that display elevated levels during the G1 phase of the cell cycle. Depletion of MIR100HG-encoded lncRNAs in human cells results in aberrant cell cycle progression without altering the levels of miRNA encoded within MIR100HG. Notably, MIR100HG interacts with HuR/ELAVL1 as well as with several HuR-target mRNAs. Further, MIR100HG-depleted cells show reduced interaction between HuR and three of its target mRNAs, indicating that MIR100HG facilitates interaction between HuR and target mRNAs. Our studies have unearthed novel roles played by a MIRHG-encoded lncRNA in regulating RNA binding protein activity, thereby underscoring the importance of determining the function of several hundreds of lnc-miRHGs that are present in human genome.

Technological Developments in lncRNA Biology.

It is estimated that more than 90% of the mammalian genome is transcribed as non-coding RNAs. Recent evidences have established that these non-coding transcripts are not junk or just transcriptional noise, but they do serve important biological purpose. One of the rapidly expanding fields of this class of transcripts is the regulatory lncRNAs, which had been a major challenge in terms of their molecular functions and mechanisms of action. The emergence of high-throughput technologies and the development in various conventional approaches have led to the expansion of the lncRNA world. The combination of multidisciplinary approaches has proven to be essential to unravel the complexity of their regulatory networks and helped establish the importance of their existence. Here, we review the current methodologies available for discovering and investigating functions of long non-coding RNAs (lncRNAs) and focus on the powerful technological advancement available to specifically address their functional importance.

ADAR2 regulates RNA stability by modifying access of decay-promoting RNA-binding proteins.

Adenosine deaminases acting on RNA (ADARs) catalyze the editing of adenosine residues to inosine (A-to-I) within RNA sequences, mostly in the introns and UTRs (un-translated regions). The significance of editing within non-coding regions of RNA is poorly understood. Here, we demonstrate that association of ADAR2 with RNA stabilizes a subset of transcripts. ADAR2 interacts with and edits the 3΄UTR of nuclear-retained Cat2 transcribed nuclear RNA (Ctn RNA). In absence of ADAR2, the abundance and half-life of Ctn RNA are significantly reduced. Furthermore, ADAR2-mediated stabilization of Ctn RNA occurred in an editing-independent manner. Unedited Ctn RNA shows enhanced interaction with the RNA-binding proteins HuR and PARN [Poly(A) specific ribonuclease deadenylase]. HuR and PARN destabilize Ctn RNA in absence of ADAR2, indicating that ADAR2 stabilizes Ctn RNA by antagonizing its degradation by PARN and HuR. Transcriptomic analysis identified other RNAs that are regulated by a similar mechanism. In summary, we identify a regulatory mechanism whereby ADAR2 enhances target RNA stability by limiting the interaction of RNA-destabilizing proteins with their cognate substrates.

Paraspeckles modulate the intranuclear distribution of paraspeckle-associated Ctn RNA.

Paraspeckles are sub-nuclear domains that are nucleated by long noncoding RNA Neat1. While interaction of protein components of paraspeckles and Neat1 is understood, there is limited information on the interaction of non-structural RNA components with paraspeckles. Here, by varying paraspeckle number and size, we investigate how paraspeckles influence the nuclear organization of their non-structural RNA component Ctn RNA. Our results show that Ctn RNA remains nuclear-retained in the absence of intact paraspeckles, suggesting that they do not regulate nuclear retention of Ctn RNA. In the absence of Neat1, Ctn RNA continues to interact with paraspeckle protein NonO to form residual nuclear foci. In addition, in the absence of Neat1-nucleated paraspeckles, a subset of Ctn RNA localizes to the perinucleolar regions. Concomitant with increase in number of paraspeckles, transcriptional reactivation resulted in increased number of paraspeckle-localized Ctn RNA foci. Similar to Neat1, proteasome inhibition altered the localization of Ctn RNA, where it formed enlarged paraspeckle-like foci. Super-resolution structured illumination microscopic analyses revealed that in paraspeckles, Ctn RNA partially co-localized with Neat1, and displayed a more heterogeneous intra-paraspeckle localization. Collectively, these results show that while paraspeckles do not influence nuclear retention of Ctn RNA, they modulate its intranuclear compartmentalization.

Knockdown of nuclear-retained long noncoding RNAs using modified DNA antisense oligonucleotides.

Long noncoding RNAs (lncRNAs) have recently emerged as important players in diverse cellular processes. Among them, a large fraction of lncRNAs are localized within cell nucleus. And several of these nuclear-retained lncRNAs have been found to regulate key nuclear processes, which brings up the requirement of effective genetic tools to explore the functions of this "dark matter" inside the nucleus. While siRNAs and shRNAs are widely used tools in loss-of-function studies, their general efficiency in depleting nuclear-retained lncRNAs is limited, due to the fact that the RNAi machinery is located mainly in the cytoplasm of mammalian cells. Here, we describe the usage of chemically modified chimeric DNA antisense oligonucleotides (ASO) in effective knockdown of nuclear-retained lncRNAs, with a focus on the detailed workflow from the design and synthesis of ASOs, to in vitro and in vivo delivery methods.

RNA fluorescence in situ hybridization in cultured mammalian cells.

It is now clear that long noncoding RNAs (lncRNAs) regulate a number of aspects of nuclear organization and gene expression. An important tool for the study of the distribution and function of lncRNAs is RNA fluorescence in situ hybridization (RNA-FISH). The protocols presented in this chapter describe this method in detail and also mention a number of critical points that must be considered when performing this technique.

PAR-CLIP analysis uncovers AUF1 impact on target RNA fate and genome integrity.

Post-transcriptional gene regulation is robustly regulated by RNA-binding proteins (RBPs). Here we describe the collection of RNAs regulated by AUF1 (AU-binding factor 1), an RBP linked to cancer, inflammation and aging. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis reveals that AUF1 primarily recognizes U-/GU-rich sequences in mRNAs and noncoding RNAs and influences target transcript fate in three main directions. First, AUF1 lowers the steady-state levels of numerous target RNAs, including long noncoding RNA NEAT1, in turn affecting the organization of nuclear paraspeckles. Second, AUF1 does not change the abundance of many target RNAs, but ribosome profiling reveals that AUF1 promotes the translation of numerous mRNAs in this group. Third, AUF1 unexpectedly enhances the steady-state levels of several target mRNAs encoding DNA-maintenance proteins. Through its actions on target RNAs, AUF1 preserves genomic integrity, in agreement with the AUF1-elicited prevention of premature cellular senescence.

Weaning of mechanically ventilated chronic obstructive pulmonary disease patients by using non-invasive positive pressure ventilation: A prospective study.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) patients frequently pose difficulty in weaning from invasive mechanical ventilation (MV). Prolonged invasive ventilation brings along various complications. Non-invasive positive pressure ventilation (NIPPV) is proposed to be a useful weaning modality in such cases. OBJECTIVE: To evaluate the usefulness of NIPPV in weaning COPD patients from invasive MV, and compare it with weaning by conventional pressure support ventilation (PSV). MATERIALS AND METHODS: For this prospective randomized controlled study, we included 50 COPD patients with type II respiratory failure requiring initial invasive MV. Upon satisfying weaning criteria and failing a t-piece weaning trial, they were randomized into two groups: Group I (25 patients) weaned by NIPPV, and group II (25 patients) weaned by conventional PSV. The groups were similar in terms of disease severity, demographic, clinical and biochemical parameters. They were compared in terms of duration of MV, weaning duration, length of intensive care unit (ICU) stay, occurrence of nosocomial pneumonia and outcome. RESULTS: Statistically significant difference was found between the two groups in terms of duration of MV, weaning duration, length of ICU stay, occurrence of nosocomial pneumonia and outcome. CONCLUSION: NIPPV appears to be a promising weaning modality for mechanically ventilated COPD patients and should be tried in resource-limited settings especially in developing countries.

Long noncoding RNA MALAT1 controls cell cycle progression by regulating the expression of oncogenic transcription factor B-MYB.

The long noncoding MALAT1 RNA is upregulated in cancer tissues and its elevated expression is associated with hyper-proliferation, but the underlying mechanism is poorly understood. We demonstrate that MALAT1 levels are regulated during normal cell cycle progression. Genome-wide transcriptome analyses in normal human diploid fibroblasts reveal that MALAT1 modulates the expression of cell cycle genes and is required for G1/S and mitotic progression. Depletion of MALAT1 leads to activation of p53 and its target genes. The cell cycle defects observed in MALAT1-depleted cells are sensitive to p53 levels, indicating that p53 is a major downstream mediator of MALAT1 activity. Furthermore, MALAT1-depleted cells display reduced expression of B-MYB (Mybl2), an oncogenic transcription factor involved in G2/M progression, due to altered binding of splicing factors on B-MYB pre-mRNA and aberrant alternative splicing. In human cells, MALAT1 promotes cellular proliferation by modulating the expression and/or pre-mRNA processing of cell cycle-regulated transcription factors. These findings provide mechanistic insights on the role of MALAT1 in regulating cellular proliferation.

SRSF1 regulates the assembly of pre-mRNA processing factors in nuclear speckles.

The mammalian cell nucleus is compartmentalized into nonmembranous subnuclear domains that regulate key nuclear functions. Nuclear speckles are subnuclear domains that contain pre-mRNA processing factors and noncoding RNAs. Many of the nuclear speckle constituents work in concert to coordinate multiple steps of gene expression, including transcription, pre-mRNA processing and mRNA transport. The mechanism that regulates the formation and maintenance of nuclear speckles in the interphase nucleus is poorly understood. In the present study, we provide evidence for the involvement of nuclear speckle resident proteins and RNA components in the organization of nuclear speckles. SR-family splicing factors and their binding partner, long noncoding metastasis-associated lung adenocarcinoma transcript 1 RNA, can nucleate the assembly of nuclear speckles in the interphase nucleus. Depletion of SRSF1 in human cells compromises the association of splicing factors to nuclear speckles and influences the levels and activity of other SR proteins. Furthermore, on a stably integrated reporter gene locus, we demonstrate the role of SRSF1 in RNA polymerase II-mediated transcription. Our results suggest that SR proteins mediate the assembly of nuclear speckles and regulate gene expression by influencing both transcriptional and posttranscriptional activities within the cell nucleus.

Malat1 is not an essential component of nuclear speckles in mice.

Malat1 is an abundant long, noncoding RNA that localizes to nuclear bodies known as nuclear speckles, which contain a distinct set of pre-mRNA processing factors. Previous studies in cell culture have demonstrated that Malat1 interacts with pre-mRNA splicing factors, including the serine- and arginine-rich (SR) family of proteins, and regulates a variety of biological processes, including cancer cell migration, synapse formation, cell cycle progression, and responses to serum stimulation. To address the physiological function of Malat1 in a living organism, we generated Malat1-knockout (KO) mice using homologous recombination. Unexpectedly, the Malat1-KO mice were viable and fertile, showing no apparent phenotypes. Nuclear speckle markers were also correctly localized in cells that lacked Malat1. However, the cellular levels of another long, noncoding RNA--Neat1--which is an architectural component of nuclear bodies known as paraspeckles, were down-regulated in a particular set of tissues and cells lacking Malat1. We propose that Malat1 is not essential in living mice maintained under normal laboratory conditions and that its function becomes apparent only in specific cell types and under particular conditions.

RNA splicing control: yet another gene regulatory role for long nuclear noncoding RNAs.

The mammalian genome harbors a large number of long non-coding RNAs (lncRNAs) that do not code for proteins, but rather they exert their function directly as RNA molecules. LncRNAs are involved in executing several vital cellular functions. They facilitate the recruitment of proteins to specific chromatin sites, ultimately regulating processes like dosage compensation and genome imprinting. LncRNAs are also known to regulate nucleocytoplasmic transport of macromolecules. A large number of the regulatory lncRNAs are retained within the cell nucleus and constitute a subclass termed nuclear-retained RNAs (nrRNAs). NrRNAs are speculated to be involved in crucial gene regulatory networks, acting as structural scaffolds of subnuclear domains. NrRNAs modulate gene expression by influencing chromatin modification, transcription and post-transcriptional gene processing. The cancer-associated Metastasis-associated lung adenocarcinoma transcript1 (MALAT1) is one such long nrRNA that regulates pre-mRNA processing in mammalian cells. Thus far, our understanding about the roles played by nrRNAs and their relevance in disease pathways is only 'a tip of an iceberg'. It will therefore be crucial to unravel the functions for the vast number of long nrRNAs, buried within the complex mine of the human genome.

A BEN-domain-containing protein associates with heterochromatin and represses transcription.

In eukaryotes, higher order chromatin structure governs crucial cellular processes including DNA replication, transcription and post-transcriptional gene regulation. Specific chromatin-interacting proteins play vital roles in the maintenance of chromatin structure. We have identified BEND3, a quadruple BEN domain-containing protein that is highly conserved amongst vertebrates. BEND3 colocalizes with HP1 and H3 trimethylated at K9 at heterochromatic regions in mammalian cells. Using an in vivo gene locus, we have been able to demonstrate that BEND3 associates with the locus only when it is heterochromatic and dissociates upon activation of transcription. Furthermore, tethering BEND3 inhibits transcription from the locus, indicating that BEND3 is involved in transcriptional repression through its interaction with histone deacetylases and Sall4, a transcription repressor. We further demonstrate that BEND3 is SUMOylated and that such modifications are essential for its role in transcriptional repression. Finally, overexpression of BEND3 causes premature chromatin condensation and extensive heterochromatinization, resulting in cell cycle arrest. Taken together, our data demonstrate the role of a novel heterochromatin-associated protein in transcriptional repression.

Nuclear organization and dynamics of 7SK RNA in regulating gene expression.

Noncoding RNAs play important roles in various aspects of gene regulation. We have identified 7SK RNA to be enriched in nuclear speckles or interchromatin granule clusters (IGCs), a subnuclear domain enriched in pre-mRNA processing factors. 7SK RNA, in association with HEXIM 1 and 2, is involved in the inhibition of transcriptional elongation by RNA polymerase II. Inhibition occurs via sequestration of the active P-TEFb kinase complex (CDK 9 and Cyclin T1/T2a/b or K) that is involved in phosphorylating the C-terminal domain of RNA polymerase II. Our results demonstrate that knock-down of 7SK RNA, by specific antisense oligonucleotides, results in the mislocalization of nuclear speckle constituents in a transcription-dependent manner, and the transcriptional up-regulation of a RNA polymerase II transcribed reporter gene locus. Furthermore, 7SK RNA transiently associates with a stably integrated reporter gene locus upon transcriptional down-regulation and its presence correlates with the efficient displacement of P-TEFb constituents from the locus. Our results suggest that 7SK RNA plays a role in modulating the available level of P-TEFb upon transcriptional down-regulation by sequestering its constituents in nuclear speckles.