LAB ROTATION 2

Mapping nascent RNA structure:optimization of the technique

Inês Martins | Laboratory rotation II | Msc in Biomedical research | Tutor: Pedro Prudêncio | Supervisor: André Gomes | MC Fonseca Lab

COTRANSCRIPTIONAL SPLICING

NUCLEAR COMPARTMENTALIZATION

DNA spatial organization impact on gene expression

Nascent RNA spatial organization

Image adapted from Guttman et al. (2021).

LONG INTRONS ARE EFFICIENTLY SPLICED

Image adapted from de Proudfoot et al. (2006).

Image adapted from Sousa-Luís et al. (2021).

HYPOTHESIS

Nascent RNA is spatially organized to facilitate efficient splicing

Image adapted from Sousa-Luís et al. (2021).

APEX2 & POINT-seq

TECHNIQUES

Image adapted from Ingolia et al. (2019).

SPATIAL LABELLING APPROACH

predictions

Input nascent RNA

Reads

Biotinylated nascent RNA

Pol II proximity regions

RT-qPCR EXPECTED OUTCOMES

SRSF1 + APEX2 (HEK293 cell line)

2 2

2 2

BCL H O

BCL H O

2 2

NASCENT RNA IS NOT DETECTED BY RT-qPCR

NASCENT RNA IS DETECTED BY RT-PCR

WHAT IS NEXT?

CHANGES in protocols

APEX2 activation

Biotinylated nascent RNA IP

Nascent RNA isolation

SuperScript III RT cDNA synthesis

qPCR for BCL

Thank you for your attention!

Special thanks to MC Fonseca's lab team, particularly André Gomes and Pedro Prudêncio

edited cell lines expressing APEX2 bound to Pol II

NASCENT RNA BIOTINYLATION + POINT-seq

biotinylation of the molecules on its vicinity

Spatial labelling APPROACH

nascent RNA isolation

nascent RNA cleavage

biotinylated nascent RNA IP using streptavidin beads

sequencing (POINT-seq)

profile of the relative distances between the nascent RNA and Pol II