Team:Heidelberg/Pumilio PPR

Pumilio and PPR
Nature's modular RBPs

Introduction

A major part of our project was to create new biological tools that would extend the capabilities of cell regulation at the protein level through the creation of novel fusion proteins connected with RNA-Linkers. On our page about RNA-protein interaction we already described basic design principles we used when designing the assembly of protein subunits through RNA linking and described the importance of two types of biological parts: RNA binding proteins (RBPs) and RNA Linkers. We described the basic viral RNA binding proteins Lambda N and MCP, we found suitable for RNA-linked protein constructs, because of their small size, simple RNA binding motif and high binding affinity to their RNA motif. Still, after having found these first two candidates for our toolbox, we wanted to dig deeper into the big world of RNA binding proteins that occur in nature and luckily found a real gem - the Pumilio Homology domain. Pumilio Homology Domains are RNA binding proteins that have a very modular structure of repeating subunits, which bind to specific ribonucleotides via two variable amino acid in every subunit. Further, in our discussions with René Inckemann, we learned about another interesting protein type - the Pentatricopeptide Repeat Proteins (PPRs), which resemble the Pumilo domains in terms of their structure and RNA binding characteristics. The big advantage when designing RNA Protein interactions with these RBPs is that they can be engineered to bind any RNA sequence. Here we present information on the design of these RBPs and provide assays that could show that these RBPs could be used for many other applications like mRNA translation control and RNA associated catalytic processes like targeted RNA degradation.

Biology and Design

Pumilio Homology Domains

Pumilio Homology Domains were first discovered in nature in Drosophila Melanogaster as part of Puf proteins. In Drosophila melanogaster they bind an RNA sequence called the Nanos response element in the maternal hunchback gene, which leads to mRNA translation control and supports the development of the thorax and head region in embryogenesisabdominal. Structurally Pumilio Homology domains resemble a half doughnut that is made up out of repeating alpha helices (Figure 1).
Figure 1: Protein Crystal Structure of native Pumilio Homology Domain
The Pumilio Homology Domain is made up out of 6 repeating subunits containing three alpha-helices and two imperfect subunits at the N- and C-Terminus containing only one subunit. The inward-looking amino acids are used to complex with RNA while the outward-looking complex interacts with different proteins pumstructure.
With the exception of the N- and C-terminal subunits, the 6 central subunits containing 3 alpha-helices have a highly similar structure. After extensive analysis of these proteins performed by multiple groups, the amino acids responsible for binding specific ribonucleobases were characterised for every subunit. Further, in 2016 the first Pumby units were created. After studying the assembly stability, the influence on cell survival and binding specificity to ribonucleotides P. Adadmala et al.pumby created a concatenated protein with multiple replicates of the 6th subunit of a native Pumilio homology domain. Additionally, they decrypted what amino acids had to be put in place at certain positions in the 6th subunit to bind each of the 4 ribonucleotides. With this information, it became possible to create concatenated RNA binding proteins of variable length, which could be designed to bind any RNA sequence (Table 1).

Table 1: Amino Acid Code for Pumby RNA binding design Pumby proteins are made out of 36 amino acid long repeating subunits. Varying the amino acids at position 16 and 20 like shown underneath lead to the complexation of the indicated ribonucleotide.

Ribonucleotide Amino Acid at position 16 Amino Acid at position 20
Adenine (A) Cysteine (C) Glutamine (Q)
Uracil (U) Asparagine (N) Glutamine (Q)
Guanine (G) Serine (S) Glutamic Acid (E)
Cytosine (C) Serine (S) Arginine (R)
To ease the process of designing concatenated Pumby proteins we added biobricks of the 6th subunit binding each of the 4 ribonucleotides to the registry:BBa_K3657000, BBa_K3657001, BBa_K3657002 and BBa_K3657003 (also see BBa_K3657004 for reference on how to construct a pumby hexamer binding the RNA motif AUAGAU). Although we got stuck in our experiment designs to pumby hexamers, it were able to show that pumbys of length of up to 18 subunits can be created with no loss of RNA binding specificity pumby.

Pentatricopeptide Repeat Proteins


The Pentatricopeptide Repeat Proteins (PPR) are a large family of modular RNA binding subunits, first discovered in Arabidopsis thaliana pprdisc. They are involved in many post-translational regulatory processes in chloroplasts, as organelle DNA lacks promoters and cellular regulation solely through transcription factors would be insufficient. They are responsible for RNA splicing, mRNA translation control and RNA editingppr.

Structurally, the PPR proteins resemble Pumilio Homology Proteins a lot. They are also made up of concatenated alpha-helices, with complex RNA with inward-looking amino acids. In contrast to the Pumby proteins, they are made up of repeating protein subunits of 35 amino acids and also their amino acid code for complexing the ribonucleotides varies from the design of the pumby subunits (Table 2) modppr.

Table 2: Amino Acid Code for PPR RNA binding design The PPR Proteins are made out of a 35 amino acid long repeating motif. Varying the amino acids 3 and 34 like shown underneath, leads to the complexation of a specific ribonucleotide in the subunit.

Ribonucleotide Amino acid at position 3 Amino acid at position 34
Adenine (A) Threonine (T) Asparagine (N)
Uracil (U) Asparagine (N) Aspartic Acid (D)
Guanine (G) Threonine (T) Aspartic Acid (D)
Cytosine (C) Asparagine (N) Serine (S)
Just like with the pumbys we created biobricks for every PPR subunit to ease the design process when constructing concatenated PPR binding proteins: BBa_K3657006, BBa_K3657007, BBa_K3657008, BBa_K3657009. Additionally, we added a N-terminal cap (BBa_K3657010) and a solubility helix that can be added to the C-terminus of the PPR to increase the solubility of the fairly unsuable PPR Proteins (BBa_K3657011). To see an example of the full construction of a PPR hexamer binding the sequence GUAGAG with added N-Cap and solubility helix see BBa_K3657013.

Experimental Validation No.1


As already discussed above both PPR and Pumilio Homology Domains are involved in mRNA translation control. We wanted to try this for ourselves and designed an assay in which we could both characterise PPR Proteins as a tunable mRNA translation inhibition tool for the regulation of protein expression and verify the correct binding of the PPR proteins to the designed target sequence (Figure 2). We designed this experiment only for PPR proteins as the suppression of mCherry translation with pumby proteins was already successfully shown with a similar experimental setup pumby.

We designed a reporter plasmid containing the RBS BBa_B0032 in combination with a super folder GFP as a reporter. Further, we designed two PPR Proteins one binding on target in the RBS with an RNA binding motif of ACACAG (BBa_K3657012) and one binding nowhere in the reporter plasmid with an RNA binding motif of GUAGAG (BBa_K3657013). We theorized that the expression of sfGFP (and thus the fluorescence intensity of our probes), should decrease in probes expressing a high amount of the on-target PPR protein, as the complexation of the PPR protein with the RBS should interfere with the translation of the mRNA. The off-target PPR protein would serve as a negative control to verify the binding specificity of our designed PPR proteins to their designated RNA motif and to ensure that the decrease of fluorescence is actually a reaction of the RBS complexation of the ON target PPR.

Cloning

To create the constructs for the experimental setup described above we had to prepare several plasmids (Table 1). As always if you would prefer to view our plasmids in the bioinformatics software of our choice you can download them here together with the constructs of our second experiment later on this page.

Table 1: Overview of Level 1 constructs for PPR mRNA inhibition assay Elements of the level 1 plasmids needed to conduct the PPR mRNA inhibition assay. All constructs were cloned with K2560034 as a terminator. All level 1 plasmids containing PPRs were cloned with K2560016 as RBS, while the reporter plasmid was cloned with BBa_K2560010 as RBS.

Name of Plasmid Promotor Coding Sequence Resistance ORI
PPR ON alpha BBa_K2560007 BBa_K3657012 BBa_K2560059 BBa_K2560046
PPR ON beta BBa_K2560017 BBa_K3657012 BBa_K2560059 BBa_K2560046
PPR ON gamma BBa_K2560014 BBa_K3657012 BBa_K2560059 BBa_K2560046
PPR ON delta BBa_K2560028 BBa_K3657012 BBa_K2560059 BBa_K2560046
PPR OFF alpha BBa_K2560007 BBa_K3657013 BBa_K2560059 BBa_K2560046
PPR OFF beta BBa_K2560017 BBa_K3657013 BBa_K2560059 BBa_K2560046
PPR OFF gamma BBa_K2560014 BBa_K3657013 BBa_K2560059 BBa_K2560046
PPR OFF delta BBa_K2560028 BBa_K3657013 BBa_K2560059 BBa_K2560046
sfGFP Reporter BBa_K2560007 BBa_K2560042 BBa_K2560125 BBa_K2560036
We created several constructs with different expression strenghts, to later be able to analyse the correlation of expressed amount of on target PPR protein to loss of sfGFP fluorescence intensity. For the exact setup of our cloning please also see our protocols, and contribution.

After the cloning every plasmid containing a PPR Protein construct would be co-transformed with the sfGFP reporter plasmid into competent E.coli. Due to limited time in the laboratory caused by the Corona pandemic we were not able to produce all plasmids on time. Therefore below we present you with our overall experiment plan as well as some preliminary results from successful constructs.

Measurement

After successful cloning of all constructs, our plan was to use a plate reader to measure the fluorescent intensity of sfGFP, which has an excitation maximum at 485 nm and an emission maximum at 510 nm. Thereby multiple replicates from the same seed culture and from different colonies picked from the agar plates after co-transformation would be measured (Figure 2).
Figure 2: Measurement layout in a black 96 well plate for mRNA translation Inhibition Assay
After co-transformation, three colonies of every construct would be picked to take variance between bacterial colonies into account. The seed-culture of the sfGFP Reporter, OFF Target PPR constructs, and ON Target PPR constructs would all be measured in four replicates per colony. Additionally, negative control with wells just containing crude culture, and empty medium, to adjust the measurement for background fluorescent, would be pipetted at the bottom of the plate.
Also see our protocols for the exact setup of fluorescent protein measurement with a plate reader. After the measurement and correction of the data to the OD600 and the subtraction of the background fluorescence of the medium, we could have plotted a curve comparing the strength of inhibition induced via the PPR ON target proteins, given different expression strengths through the Anderson promoters. We also would compare the performance between the ON target PPR and OFF target PPR.

Preliminary results

Because of Covid-19, we were unfortunately only able to start working in the wet lab at the beginning of September. We were therefore not able to construct all of the plasmids needed to conduct proper measurements of the designed experiments. We were only able to conduct one fluorescent measurement with the PPR ON delta, PPR OFF delta and sfGFP Reporter (Figure 5), as all other plasmid level constructs were partly missing.
Figure 5: Boxplot of fluorescent intensities from mRNA translation inhibition assay
The measurements were conducted with the sfGFP Reporter under a BBa_J23100, and ON and OFF target PPRs under a weak Anderson promoter BBa_J23115. Boxplots were created from 24 measurements per sample originating from 3 colonies per sample.
The boxplot suggests that there is no significant difference in sfGFP expression between the positive probe containing solely sfGFP and the sample containing the ON target PPR. The negative control expressing an OFF target PPRs exhibited even a lower fluorescence. Although these results suggest that there does not seem to be an inhibition originating from the PPR proteins, we would not already call them off. The Anderson promoter BBa_J23100 expresses proteins 6.4 times stronger than the BBa_J23115 expressing the PPR proteins. Additionally, the PPR proteins were cloned into a plasmid containing the p15A ORI, which has a lower replication number than the ColE1 ORI that the sfGFP Reporter plasmid contains. We conclude that we would have definitely carried on with this experiment and tried to measure other probes designed by us like the PPR ON target alpha level 1 plasmid, to finally determine whether the designed PPR proteins actually do not interfere with the translation of the sfGFP reporter mRNA.

Biology and Design No.2

Now, this was fairly exciting already, was it not? We now can work with RNA binding proteins that can bind any sequence that you design them to bind and created, next to our split-GFP assay, another way of validating whether RNA binding proteins which actually bind to their RNA motif. But we did not want to stop there. We mentioned earlier that the PPR proteins are involved in many different catalytic processes associated with RNA. In these cases, they are often made up of the concatenated alpha helices that bind specific ribonucleotides and are followed by a catalytically active protein subunit. Like this, PPR proteins are involved in the polycistronic translation of mRNA in chloroplasts, make complexes with proteinaceous ribonuclease P to process tRNAs or bind to homing endonucleases, which are responsible for DNA mobility between organelles in eukaryotes ppr. We further were reminded of the RNAse L, a human ribonuclease that is activated when a human cell shifts into a metabolic state of viral defence rnasel. The RNAse L starts to rapidly degrade single-stranded viral RNA in the cell. We wanted to take a shot at designing a catalytical PPR protein, to validate whether a combination of PPR and a catalytical protein would specifically target RNA structures matching the RNA motif, and to make a first step towards designing catalytical PPR proteins, may they exist in nature already or not.

Experimental Validation No.2

Inspired by the shortly mentioned RNAse L we wanted to try to create a universal ribonuclease, by connecting a PPR protein to an unspecific ribonuclease, hoping that such a complex would primarily target structures containing the RNA binding motif of the PPR protein. We decided to use the ribonuclease A, as this enzyme can cut basically any sequence, with the only restriction of having to cut next to a pyrimidine pyrimidine. But we could not simply have conducted the mRNA translation inhibition experiment again, because if this assay would turn out positive, the fluorescence intensity decreasing could simply be explained by the translation inhibition of the RNA bound PPR protein and not necessarily by the degradation of the mRNA. We thus decided to choose another form of reporter molecule - an aptamer. Aptamers are short molecules made up of nucleic acid that can bind to small molecules. We decided to use Broccoli, which is an aptamer complexing DFHBI, a chromophore that emits green fluorescence when stabilized by broccoli broccoli. Because aptamers do not have to be translated, the risk of fluorescence inhibition by the PPR Protein would be minimized. Instead, we hoped to see, that if the PPR protein binds to a recognition sequence in the broccoli aptamer the ribonuclease would primarily degrade the broccoli aptamer, leading to a loss of fluorescence.

Broccoli Reengineering

When designing this assay we stumbled over an unforeseen problem. PPR proteins normally bind single-stranded RNA. The structure of the Broccoli Aptamer, however, has no regions at which a PPR hexamer could fully bind. To make matters worse the original sequence of the Broccoli aptamer has a reverse BsmbI cut site. We could therefore not have cloned with the Marburg Collection cloning standards, because the broccoli sequence simply would have been cut in half. Because of this, we had to redesign the sequence of the Broccoli Aptamer (Figure 3).
Figure 3: Scheme of original and RNA secondary structure of the redesigned Broccoli Aptamer
On the left a scheme of the original broccoli sequence. The Red Line indicates the reverse BsmbI cut site GCAGAG. On the right, the secondary structure of the newly designed broccoli, without the BsmbI cut site and with an added single-stranded RNA sequence in which the PPR Protein could bind.
We redesigned the broccoli aptamer using Designsimple, a software tool we already used to design the hammerhead ribozymes for our RNA linkers. It took the secondary structure of broccoli as input and was given sequence constraints of specific nucleotides that were essential for the complexation of DFHBI and therefore could not be altered, and generated a new sequence while keeping the secondary structure of the original broccoli. We created three versions of the redesigned broccoli: one only being depleted of the BsmbI cut site (BBa_K3657016, one being depleted of the BsmbI cut site and containing the added hair loop for improved binding of the PPR Protein (BBa_K3657017 and a dimeric version of the broccoli with the added loop (BBa_K3657018).

Cloning

We now had all the parts we needed to conduct the experiment. First, we again designed two PPR proteins, one ON Target PPR Protein binding the RNA sequence GUAGAG in the loop or stem region of the broccoli aptamer(BBa_K3657013), and one OFF target PPR Protein binding the RNA sequence ACACAG which cannot be found in the broccoli aptamers. (BBa_K3657012). Additionally to the PPR-Ribonuclease A constructs we designed Level one plasmids, only containing a Ribonuclease or only containing the PPR proteins, in order to compare the effectiveness in decreasing fluorescence intensity of the PPR-Ribonuclease complex to the subunits on their own. As always if you would rather like to view our plasmid construct in the bioinformatics software of your choice you can download our plasmid constructs here.

Table 4: Overview of Level 1 plasmids for the PPR-Ribonuclease experiment Overview of the elements of level 1 plasmid constructs needed to conduct the PPR-Ribonuclease A experiment. All parts were cloned with BBa_K2560034 as terminator. All Broccolis were cloned with BBa_K2560007 as promotor. The PPR or ribonuclease constructs were cloned with different Anderson promoters to relate the effect of expression frequency better to the decrease of fluorescence intensity in the measurement. All PPR and Ribonuclease constructs were cloned with BBa_K2560016 as RBS. No RBS was cloned into the Broccoli constructs to avoid translation, instead, the Broccolis were given 5' Mo-Clo Overhangs of the RBS to bind directly to the promoter.

Name of Plasmids Coding Sequence Resistance ORI
PPR-Rib. ON Target BBa_K3657021 BBa_K2560059 BBa_K2560046
PPR-Rib. OFF Target BBa_K3657054 BBa_K2560059 BBa_K2560046
PPR ON BBa_K3657012 BBa_K3657012 BBa_K2560046
PPR OFF BBa_K3657013 BBa_K3657012 BBa_K2560046
Ribonuclease A BBa_K3657019 BBa_K3657012 BBa_K2560046
Broccoli no BsmbI BBa_K3657016 BBa_K2560125 BBa_K2560036
Broccoli with Loop BBa_K3657017 BBa_K2560125 BBa_K2560036
Broccoli 2x with Loop BBa_K3657018 BBa_K2560125 BBa_K2560036
After successfull creation of the level 1 plasmids. Every PPR or ribonuclease A construct would be co-transformed with a broccoli plasmid into competent E.coli.

Measurement

First, we would have to assess whether our newly designed broccolis actually work as intended. We therefore first would only transform cells with the plasmids of broccolis and measure them with a plate reader (see our protocols for an exact description of the fluorescence measurement of broccolis).
After the validation of broccoli's function, we would proceed to measure the probes of PPR and ribonuclease constructs and their effect on the broccoli aptamers (Figure 4).
Figure 4: Measurement layout in a black 96 well plate for PPR Ribonuclease experiment
3 colonies per construct would be picked after co-transformation and growth on agar plates. Of every resulting seed culture, 4 replicates would be measured. We would measure a seed culture only transformed with the broccoli plasmid, seed cultures containing On and Off-target PPR Ribonuclease constructs and seed cultures of constructs containing only the PPR Proteins or only the Ribonucleases to compare the performance of the single subunit to the performance of the PPR-Ribonuclease construct.
For the exact measurement procedures with bacteria containing broccoli, see our protocols page. After the measurement of the data and the normalisation of the values to the background fluorescence and the OD600 of the well, we would proceed to compare the performance of the PPR-Ribunuclease construct to the other constructs by calculating the ratios of the measured performance between the fluorescence intensities of the construct to the fluorescent intensities of the positive control. Additionally, to the measurement of fluorescence, we would also conduct a cell growth assay as we suspect that ribonucleases A Complexes may have a high metabolic burden, as the ribonuclease would probably not only degrade the RNA containing the PPR binding motif but have an off-target activity that may interfere with a lot of protein expression.

References