Identifying putative functions of long non-coding RNAs in trees

You worked already during your master’s thesis together with Nathaniel Street on a bioinformatics project. What convinced you to continue with a PhD in his group?

Camilla Canovi: Yes, I was doing an Erasmus traineeship together with Nathaniel Street. I did my Master’s in Italy. My professor there knew Nathaniel and send me here. We worked well together and to know him and his group made it very easy for me to say yes when he offered me the PhD project. It made it also easier for me to tell him that I do not want to spend all my days in front of a computer but would like to do also things in the lab. I know myself and that I need some variety to keep my motivation up. So, we adjusted the study plan accordingly. I really appreciate that Nathaniel gave me this freedom and the possibility to take my own choices during my PhD.

You studied long non-coding RNAs in Norway spruce and aspen. What are long non-coding RNAs?

Camilla Canovi: There is a lot of DNA that is transcribed into RNA but does not contain information for a protein. All such RNA is called non-coding RNA. Some of them are more known such as microRNAs and small interfering RNAs because they have been discovered earlier. Those ones usually have a length of below 200 base pairs and some of their functions are well studied already. All non-coding RNAs that are longer than 200 base pairs are defined as long non-coding RNAs. This group is much more diverse. The sequences often differ a lot even between relatively close related species such as spruce and pine. Sometimes only the location in the genome is conserved between species but not the DNA sequence itself which made it very difficult to study them.

Long non-coding RNAs are activated when plants are exposed to stress

Why is it important to study long non-coding RNAs and what are their functions?

Camilla Canovi: Not only the structure but also the functions of long non-coding RNAs can be very different. They can regulate gene activity in many different ways, or they can influence how accessible a certain gene is by modifying the packing of the DNA double strand. They can also serve as decoys that fish out microRNAs so that those ones are not interfering with the expression of a gene anymore. In plants, they seem to be mostly activated when the plants are exposed to stress. In humans, they are a bit better studied because they can serve as marker for cancer. However, the high diversity in function and appearance makes it difficult to study long non-coding RNAs and there is not much done yet with respect to trees like spruce and aspen.

What do you consider as the major outcome of your thesis?

Camilla Canovi: To identify long non-coding RNAs with bioinformatics analyses, we need to first define parameters which is a challenge with such a diversity in appearance. I focused on a certain group of long non-coding RNAs that do not overlap with any gene but are located only in regions between genes and developed a bioinformatics pipeline to identify them in plants. I have applied this pipeline on spruce and aspen to look for this subgroup of long non-coding RNAs, but it can be also used on other plants and to search for other types of long non-coding RNAs by modifying some parameters.

In a next step, I constructed a co-expression network to see which genes are active at the same time as the identified long non-coding RNAs. Then, I checked in which biological processes those genes were involved and assumed that the long non-coding RNA is involved in the same processes as the genes, like for example photosynthesis or leaf development. And finally, I wanted to check if our predictions were correct and modified aspen trees using the CRISPR-Cas9 technology to remove a putative long non-coding RNA. This work is still in process, but I managed to get the first modified trees and some of them are really promising. That is very exciting!

The first modified aspen trees look really promising

Where there any results that you did not expect?

Camilla Canovi: Many things were unknown when I started, and it was not clear what to expect, especially when trying to modify long non-coding RNAs in aspen with CRISPR-Cas9. There are a few studies in Arabidopsis but not for aspen or spruce as far as I know. I focused on long non-coding RNAs that are involved in leaf development and tried to cut them out. We did not expect that this would work right away but when I tested the first modified aspen trees, it looked like our strategy worked out. There are still many more tests to do but I was very happy to see that.

Your title starts with “Tackling a genomic abyss”. Did you face any other than the “genomic abyss” during your PhD?

Camilla Canovi: When I was about to start the functional validation of some of the identified long non-coding RNAs, I discovered that we had an error in the pipeline. The programme that should make sure to choose only regions in between genes did not work properly. So, we had to fix that which costed me quite some time. However, I was very glad that I realized it before starting with modifying aspen trees which takes even more time.

And then, there was of course the Covid-19 pandemic, and I was somehow stuck here for one year and nine months which was a bit too long. I did not want to go home where my grandmothers were waiting to see me and risk that they get infected. That felt quite long but luckily, I had good friends here.

What are you planning to do now?

Camilla Canovi: My contract lasts until January which gives me time to finish the last things. But first, I will go to Namibia for vacation for ten days to see a desert. I have never seen one before and I am curious about that. Then, I would like to change air and go back to Italy and move away from academia. I would like to try working in industry and have started to look for companies in Northern Italy now. I will see how it goes.