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RNA localization in bacteria

Periodic Reporting for period 1 - NucLoc (RNA localization in bacteria)

Reporting period: 2018-09-01 to 2020-08-31

In eukaryotic cells mRNAs can be localized to specific places within a cell to achieve protein synthesis with spacial and temporal control. Recently, I and others have shown that bacteria can also target mRNAs to specific regions of the cell.

Problem being addressed-
Previous mRNA localization studies in bacteria were primarily restricted to a limited number of mRNAs, and due to the serendipitous nature of these discoveries and conflicting results, it is not clear how widespread mRNA localization is in bacterial cells and what conserved mechanisms are playing a role. To investigate this, I will assess the localization of mRNAs coding for different classes of localized proteins in the model bacterium Bacillus subtilis.


Overall objectives-
1) To develop single molecule RNA FISH (smFISH) to visualize mRNAs in Bacillus subtilis
2) Develop a novel next generation sequencing based approach to identify all the mRNAs localized at the bacterial membrane
3) To investigate the role of protein translation, genome location and cell morphology on bacterial mRNAs localization


This project is designed provide a unique insight into the scale and mechanisms of mRNA localization in bacteria.
a) Single molecule RNA FISH in Bacillus subtilis
-smFISH was established and tested on several classes of mRNAs. Firstly, several localized proteins (cell division, polar, cytoskeletal proteins) of B. subtilis were fused to GFP and checked for their correct localization within the cell. Multiple fluorescent DNA probes were designed to bind and detect GFP mRNA. Fusion of GFP to the desired targets therefore allowed us visualize individual mRNAs for any GFP tagged gene using the same set of fluorescent probes. However, the mRNA localization of several target proteins did not co-localize with their respective mRNAs. The results of smFISH were disseminated at the general meeting of Bacterial Cell biology labs at the host Institute (SILS, University of Amsterdam) and 1st year bachelor students of microbiology course at University of Amsterdam as a lecture.

b) Mem-seq: A novel technique to identify membrane proximal RNAs
-A new cross-linking based approach was developed to fix the localized mRNA to the bacterial membrane. After several iterations using different fixative agents, I was able to optimize the fixation and extraction of localized mRNAs from purified bacterial membrane. Next generation sequencing analysis of the isolated RNA revealed enrichment of several mRNAs encoding for inner membrane proteins in the membrane fraction compared to the total RNA analysis. The results from this technique were presented at the Amsterdam microbiology lecture series (AMZA 2018) and annual day of the host Institute (SILS day 2018).
-The Mem-seq technique is expected to result in a publication after validation of its primary results by general molecular biology approaches.

-This technique will allow researchers to get a global overview of mRNA localization in bacteria in a single experiment. It is also expected that derivations of this technique will provide information on mRNA localized at the membrane-bound organelles in Eukaryotes.