Introduction

I am going to start by talking about ... tRNA.. so my thought is, ... relating to or up to now. So my lab has been working on this for the last few years on synthetic yeas genome consortium, Sc2.0. This yeast project started when I was a postdoc a few years ago, this is a great consortium that I have been privileged to work with.

The tRNA neochromosome is where we move all the tRNA genes into the new chromosome. What we have done is moved all tRNA genes into this chromosome. Daniel Schindler and Roy Walker. It's 181 kb. It's a hybrid of about 9 yeast species. 10% seuence derived from Sacchromyces cerevisiaie.

Design the basics

I am going to talk about design basics. This is a chromosome with... so the first thing we did is we look for centromeres. Site of kinetochore assembly during cell division. Yeast has poiont centromeres, they are small about 125 bp, region with a conserved sequence on chromosome 6. Regional centromeres are large and epigenetically defined.

We're lucky that we have this point centromere. We need replication. ARS elemens (autonomously replicating sequences) are 11 bp to 17 bp T-rich core that is bound by origin recognition complex (ORC) throughout cell cycle. Eukaryotes have many origins, not all active. Replication origins are loaded with double-hexaers of MCM2-7 during S-phase, these are activated to form the core of the replicative helicase.

We put in roughly 40 kb each. The third thing we need is telomere, which will protect the chromosome from shortening and chromosome fusion. Telomeres in yeast consist of approximately 300 bp tandem TG-rich repeats. End replication problem: telomeres gradually shorten after every round of cell division, Okazaki fragments requires RNA primers attaching on the lagging strand.

We also look at Ter sites. Ter sites exist in rDNA locus to protect against replications stress. Fob1 is the primary replication fork blocking (RFB) protein. In all observed cases, Ter sites ensure that the replication for kalways move in the same direction as that of transcription.

Hierarchical design of tRNA neochromosomes

We took a few hundred tRNA genes-- they are 79-91 base pairs each. We add 500 bp and 40 bp region for the ... so this is remarkable each species has no transposon at all. To answer an earlier question about the basis... high gene... so we design this roughly 600 bp and then we trying to.. pair it with a ... we condition these. We can modulate this with..

tRNA neochromosome, tRNA mega array about 20 kb, tRNA array, tRNA casette.

Different chromosomes have different numbers of tRNA genes. ARS elements from Candida glabrate (from Conrad Niduszynski, Oxford).

Single copy tRNA KO: phenotypic characterization

Spotted onto SC-Leu (triplicate), SUP61, TRT2 TRR4, pRS415.

Genome-wide tRNA profiling

Traditional RNAseq methods are dififcult, tRNAs hae a tight secondary and tertiary structure. Hard-stop tRNA modifications inhibit reverse-transcriptase.

tRNA sequencing solutions: .. use a thermostable ...

Conclusions

tRNA neochromsome expresses very well. Pol 3 occupancy on the neo chromosome is like wildtype. Alternative splicing is not required for tRNA. Yeast has not many introns, bu we take them out. We take out all the introns, right. The other thing we found, quite interesting to mention, when we finish the chromosome and have a .. without any tRNA ...

David Tollervey, UoE

Lars Steinmetz, EMBL

Transcriptome profiling

Seuencing performed by Jackie Yun Wang at BGI on 138 kb neochromosome.

Amino acid biosynthesis up-regulated in neo strain, however translation-related functions were down-regulated.

Data inversely correlates to that of SynII (restored once chromosome 2 tRNA array added)

Identifying moonlighting jobs of SEN complex

The Sen complex is an endonuclease responsible for splicing pre-tRNA introns

Interestingly located on the surface of mitochondria (unknonw purpose)

All four SEN subunutis are essential (Sen2, Sen34, Sen15, and Sen54)

May have hidden functions in addition to tRNA splicing (moonilghting mRNA processing?)

Hypothesis: As we removed introns from the neochromosom, can we render the SEN complex non-essential?

tRNAs and the nucleus

We're also looking into this, tRNA genes likely contribute to genome structure, but literature not clear + contradictory. Evidence suggests nuclear clustering, but may congregate near centromere or nuclear pore complex.

Roy Walker
Wei Lu
Chantai Shen
Paulina Kangowska
Emily Scher
Jamie Auxillos
Katarina Johnson
Andrea Martella
Dariusz Abramczyk
Eva Garcia-Ruiz
Daniel Schindler
Aileen Greig

Q&A

Q: ...

A: ...