Identification and comparative analysis of the β-actin mRNA interactome by RNA-proximity labeling in mouse embryonic fibroblast

The temporal and spatial expression of genes is required for maintaining cellular asymmetry, proper embryonic development, neuronal function, and cell fate. In mouse embryonic fibroblasts (MEFs) this cellular asymmetry is generated by localizing various cellular mRNAs to the protrusions (lamellipodia/filopodia). Among those mRNAs, β- actin mRNA plays a major role in defining cellular asymmetry by its localization to the cell periphery. Upon mRNA localization and translation, β-actin protein helps the cells to respond to extracellular cues and to move during extracellular matrix remodeling to maintain tissue homeostasis and tissue repair, traversing changes in local tissue environments as needed in tissue degradation, repair or regeneration. Under normal trophic conditions, the localization of β-actin mRNA to the cellular protrusions of fibroblasts or growth cones in neurons is regulated by a cis-acting localization element or localization signal known as zipcode (in case of β-actin, this is a 54 nt long sequence in the 3’UTR of the β-actin mRNA adjacent to its stop codon) together with trans-acting factors, mainly RNA-binding proteins (RBPs) that either bind directly to the zipcode or regulate the binding of other RBPs to it. In the case of the motor-driven movement of these localized mRNAs, such RNA-protein complexes are then tethered to molecular motors such as kinesin, dynein, or myosin, to form transport or locasome complexes. Thus, messenger ribonucleoprotein particles (mRNPs) that act as functional units not only contain the information for an encoded polypeptide but also determine the precise spatio-temporal regulation of its translation, thereby facilitating the correct subcellular localization of the translation product. It has been shown that the localization of β-actin mRNA is dependent on the binding of the zipcode-binding protein ZBP1 (an RBP of the conserved VICKZ RNA-binding protein family) to its cognate site present in the 3’UTR of the mRNA. ZBP1 (also called IGF2BP1 or IMP1) interacts with the zipcode via two K-homology (KH) RNA-binding domains by RNA looping mechanism and is required for β-actin mRNA localization in migrating cells including fibroblasts and neurons. In addition, in fibroblasts, it is also known that it controls the translation of β-actin by blocking the assembly of ribosomes at the start codon. Apart from ZBP1, the RBPs IGF2BP2, RACK, KHDRBS1/Sam68, and FMR1 play important roles during the localization of the mRNA. To obtain a complete picture of the associated proteome of any mRNA has been challenging. The high throughput methods available so far (like CLIP, MS2 pull down) mainly fail in the identification of indirect or transient interactors of specific RNAs. To solve these issues, I applied a BioID method where a protein of interest is fused to a mutant version of the E.coli biotin ligase BirA (BirA*), which biotinylates accessible lysine residues of proteins present in its vicinity. After cell lysis, biotinylated proteins can be isolated by streptavidin affinity purification and identified using standard mass spectrometry techniques. In this thesis, I report that tethering of BirA* to a specific localized, MS2- tagged mRNA does not only allow the identification of its associated proteins but can also be used to probe the environment of this mRNA. This approach allows, with high confidence, to identify novel functional β-actin interactors like FUBP3/MARTA2, STAU1, and STAU2. FUBP3 is an RBP from the conserved FUBP family of proteins. FUBP3 shown to mediate the dendritic targeting of MAP2 mRNA in neurons. In this thesis, I report FUBP3 to bind to and facilitate localization of β-actin mRNA to fibroblast protrusions. By immunoprecipitation and in vitro binding assays, I could demonstrate that it binds 460 nt downstream of the stop codon in the β-actin 3’ UTR and participates in the localization of the mRNA to the cellular protrusions. Apart from BirA* I also applied direct MS2-MCP pull-down APEX2-mediated biotin labeling of beta-actin associated proteins and compared the obtained datasets of the proteins that bind to the β-actin mRNA directly or via transient interactions. The established method convincingly shows 1. Additional proteins which could be a part of the β-actin localization complex. Amongst all these proteins, FUBP3 has shown to be a part of the β-actin localization complex for the first time. 2. FUBP3 to bind to downstream of the localization element at the 3’UTR of β-actin mRNA and is essential for the localization of β-actin mRNAs at the protrusions of fibroblasts. 3. Comparison of the β-actin proteome under serum-starved and unstarved conditions and the difference between the associated RNA interacting proteome under these two conditions

A rare case of cervical ectopic: potential life-threatening condition managed conservatively with uterine artery embolisation and Methotrexate

A case of cervical pregnancy managed successfully in Medical College, Kolkata by injection Methotrexate and uterine artery embolization. Cervix is a rare implantation site for ectopic pregnancy. Either during surgical management should be carefully considered due to the risk of severe hemorrhage. A 33 years old patient (P2+4 with 1 living issue) with USG diagnosed 6 weeks cervical pregnancy was admitted in Gynaecology and Obstetrics department of Medical College, Kolkata with slight bleeding per vaginum and pain abdomen. USG was repeated along with beta hCG quantification and other routine investigations. Following admission, the bleeding and pain subsided. Patient was counselled regarding the prognosis and management options available. The patient was desirous to preserve her fertility and as the patient was hemodynamically stable with low initial beta hCG of 5200 mIU/ml, we opted for a medical treatment with MTX and uterine artery embolization. Following treatment with 3 doses of weekly Methotrexate (50 mg/m2 each i.m.) and UAE the beta hCG reduced significantly. The patient was discharged with advice of weekly follow up with beta hCG report. Within 2 months of follow-up, ß-hCG level was <10 mIU/mL with TVS showing normal cervical canal and empty uterine cavity

Vesicular transport of a ribonucleoprotein to mitochondria

Intracellular trafficking of viruses and proteins commonly occurs via the early endosome in a process involving Rab5. The RNA Import Complex (RIC)-RNA complex is taken up by mammalian cells and targeted to mitochondria. Through RNA interference, it was shown that mito-targeting of the ribonucleoprotein (RNP) was dependent on caveolin 1 (Cav1), dynamin 2, Filamin A and NSF. Although a minor fraction of the RNP was transported to endosomes in a Rab5- dependent manner, mito-targeting was independent of Rab5 or other endosomal proteins, suggesting that endosomal uptake and mitotargeting occur independently. Sequential immunoprecipitation of the cytosolic vesicles showed the sorting of the RNP away fromCav1 in a process that was independent of the endosomal effector EEA1 but sensitive to nocodazole. However, the RNP was in two types of vesicle with or without Cav1, with membrane-bound, asymmetrically orientated RIC and entrapped RNA, but no endosomal components, suggesting vesicular sorting rather than escape of free RNP fromendosomes. In vitro, RNP was directly transferred from the Type 2 vesicles to mitochondria. Live-cell imaging captured spherical Cav12 RNP vesicles emerging from the fission of large Cav+ particles. Thus, RNP appears to traffic by a different route than the classical Rab5- dependent pathway of viral transpor

Impact of environmental factors on maintaining water quality of Bakreswar reservoir, India

Reservoirs and dams are engineered systems designed to serve purposes like supply of drinking water as well as other commercial and industrial use. A thorough assessment of water quality for these systems is thus necessary. The present study is carried out at Bakreswar reservoir, in Birbhum district, which was created by the dam, built on Bakreswar River. The major purpose of the reservoir is the supply of drinking water to the surrounding villages and Bakreswar Thermal Power Station. Water samples were collected fortnightly from three different stations of the reservoir. Physical and chemical factors like dissolved oxygen, atmospheric temperature, pH, conductivity, salinity, solar radiation, water temperature, alkalinity, hardness, chloride, productivity etc. were analysed using standard procedure. Abundance data is calculated for four major groups of zooplanktons (Cladocera, Copepoda, Ostracoda, and Rotifera) with the software PAST 2.1. Multivariate statistical analysis like PCA, hierarchical cluster and CCA are performed in order to predict the temporal variation in the water quality factors using SPSS 20. Distinct seasonal variation was found for environmental factors and zooplankton groups. Bakreswar reservoir has good assemblage of zooplankton and distinct temporal variation of environmental factors and its association with zooplankton predicts water quality condition. These results could help in formulating proper strategies for advanced water quality management and conservation of reservoir ecosystem. Key elements for growth and sustenance of the system can then be evaluated and this knowledge can be further applied for management purposes

Engagement of Components of DNA-Break Repair Complex and NFκB in Hsp70A1A Transcription Upregulation by Heat Shock.

An involvement of components of DNA-break repair (DBR) complex including DNA-dependent protein kinase (DNA-PK) and poly-ADP-ribose polymerase 1 (PARP-1) in transcription regulation in response to distinct cellular signalling has been revealed by different laboratories. Here, we explored the involvement of DNA-PK and PARP-1 in the heat shock induced transcription of Hsp70A1A. We find that inhibition of both the catalytic subunit of DNA-PK (DNA-PKc), and Ku70, a regulatory subunit of DNA-PK holo-enzyme compromises transcription of Hsp70A1A under heat shock treatment. In immunoprecipitation based experiments we find that Ku70 or DNA-PK holoenzyme associates with NFκB. This NFκB associated complex also carries PARP-1. Downregulation of both NFκB and PARP-1 compromises Hsp70A1A transcription induced by heat shock treatment. Alteration of three bases by site directed mutagenesis within the consensus κB sequence motif identified on the promoter affected inducibility of Hsp70A1A transcription by heat shock treatment. These results suggest that NFκB engaged with the κB motif on the promoter cooperates in Hsp70A1A activation under heat shock in human cells as part of a DBR complex including DNA-PK and PARP-1

A rare case of cervical ectopic: potential life-threatening condition managed conservatively with uterine artery embolisation and Methotrexate

A case of cervical pregnancy managed successfully in Medical College, Kolkata by injection Methotrexate and uterine artery embolization. Cervix is a rare implantation site for ectopic pregnancy. Either during surgical management should be carefully considered due to the risk of severe hemorrhage. A 33 years old patient (P2+4 with 1 living issue) with USG diagnosed 6 weeks cervical pregnancy was admitted in Gynaecology and Obstetrics department of Medical College, Kolkata with slight bleeding per vaginum and pain abdomen. USG was repeated along with beta hCG quantification and other routine investigations. Following admission, the bleeding and pain subsided. Patient was counselled regarding the prognosis and management options available. The patient was desirous to preserve her fertility and as the patient was hemodynamically stable with low initial beta hCG of 5200 mIU/ml, we opted for a medical treatment with MTX and uterine artery embolization. Following treatment with 3 doses of weekly Methotrexate (50 mg/m2 each i.m.) and UAE the beta hCG reduced significantly. The patient was discharged with advice of weekly follow up with beta hCG report. Within 2 months of follow-up, ß-hCG level was &lt;10 mIU/mL with TVS showing normal cervical canal and empty uterine cavity

CLIP and complementary methods

Hafner M, Katsantoni M, Köster T, et al. CLIP and complementary methods. Nature Reviews Methods Primers. 2021;1(1): 20.RNA molecules start assembling into ribonucleoprotein (RNP) complexes during transcription. Dynamic RNP assembly, largely directed by cis-acting elements on the RNA, coordinates all processes in which the RNA is involved. To identify the sites bound by a specific RNA-binding protein on endogenous RNAs, cross-linking and immunoprecipitation (CLIP) and complementary, proximity-based methods have been developed. In this Primer, we discuss the main variants of these protein-centric methods and the strategies for their optimization and quality assessment, as well as RNA-centric methods that identify the protein partners of a specific RNA. We summarize the main challenges of computational CLIP data analysis, how to handle various sources of background and how to identify functionally relevant binding regions. We outline the various applications of CLIP and available databases for data sharing. We discuss the prospect of integrating data obtained by CLIP with complementary methods to gain a comprehensive view of RNP assembly and remodelling, unravel the spatial and temporal dynamics of RNPs in specific cell types and subcellular compartments and understand how defects in RNPs can lead to disease. Finally, we present open questions in the field and give directions for further development and applications

NFκB modulates Hsp70A1A promoter activity under heat shock.

<p>A) Schematic showing the regions of Hsp70A1A promoter fragments cloned upstream of renila luciferase reporter (Rluc). The locations of heat shock elements (dHSE, distal heat shock element; pHSE, proximal heat shock element), NF-Y and κB consensus sequence (κBc) and a less conserved κB (?) site are indicated with respect to transcription start site (+1, an arrow). The direction of the arrow indicates the direction of transcription. B) RT-PCR assay with transcripts prepared from HEK293 cells 48 h post transfection with the constructs shown in panel ‘A’. Representative ethidium bromide stained agarose gels showing relative levels of indicated transcripts. C) Estimation of the band intensities of luciferase cDNA vs those of GFP and GAPDH as the internal control. D) RT-PCR assay with transcripts prepared from HEK293 cells 48 h posttransfection with the constructs indicated (also shown in panel A, constructs #3 and #3(M)). Representative ethidium bromide stained agarose gels showing relative levels of transcripts as indicated. E) Estimation of the band intensities of lucifearse cDNA vs those of GFP and GAPDH as the internal control. HS, heat shock.</p

DNA-PK holoenzyme is required for induction of Hsp70A1A transcription in response to heat shock (HS).

<p>cDNAs prepared from cells pre-treated with Ku70 specific (siKu70), or scrambled siRNA were subjected to PCR to determine expression levels of indicated genes. A) Representative ethidium bromide stained agarose gels indicating the relative levels of Ku70 and Hsp70A1A transcripts. B) The abundance of the indicated transcripts/cDNAs (as indicated in panel A) were estimated by RT-qPCR normalized with normalized with β-actin level. One-way ANOVA with Turkey’s post-test was used to analyse the data where ***p < 0.001. C) Immunoblot showing downregulation of Ku70 protein in whole cell lysate isolated from HeLa cells pre-treated with siKu70 or scramble siRNA. Fold change shown was estimated by densitometric scanning of intensities of bands of Ku70 vs β-actin. D) Representative ethidium bromide stained agarose gels showing the levels of transcripts isolated from cells pre-treated with siKu70, or shDNA-PKc or both siKu70 and shDNA-PKc simultaneously determined by RT-PCR assay. E) Estimation of the intensities of bands shown in panel D through densitometric scanning. The β-actin level was used as the loading control (bottom panel). One-way ANOVA with Turkey’s post-test was used to analyse the data where ***p < 0.001.</p