SR Proteins
From Figure 1 of: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2965536/

Figure 1 shows the various protein / gene symbol names from arabidopsis for the SR family.

To get a list of these genes, the image was copied into Google Images and translated into text.
The text was then copied into a Google Sheet and edited.

SR splicing factors Sheet:
https://docs.google.com/spreadsheets/d/1ScfcUkmf74G6eOoGGDV-3ZpQ0iqbOiVqF78SyEiPSoM/edit?usp=sharing

*A few of the entries were incorrect in the OCR step from Google lens / images and were manually corrected.

Now we have a list of arabid gene id’s! (and rice too)

Finding the relevant GO terms
To find the relevant GO terms, each of the 17 arabid ID’s were entered into TAIR’s online tool for GO term retrieval (Via PANTHER’s DB)

GO TOOL:
https://www.arabidopsis.org/tools/go_term_enrichment.jsp

Each ID found a suitable GO term. The GO term, and the link to the Panther result were recorded in the SR Splicing factors Google sheet.

( https://docs.google.com/spreadsheets/d/1ScfcUkmf74G6eOoGGDV-3ZpQ0iqbOiVqF78SyEiPSoM/edit?usp=sharing
)

Tomato genes that match…..
For 8 of the 17 Arabid SR genes, there is an already identified Sol Lyco ID that has been made (NOT Genome version SL5). See column SolyID.

These are included in the spread sheet. 9 do not have a match.

Finding matches via Reciprocal Best Hit Blasts
Goal: to see what high fidelity matches we can get by blasting the 17 aribid genes against the tomato SL5 protein sequences.
Pull the arabid sequences from TAIR and save as fasta on the cluster
Blast all soly protein sequences to these 17 seq. (blastx)
Blast the 17 against the soly proteins (tblastn).
Identify best matches somehow.
Identify 1:1, 1tomany and manyto1.

Step 1:
The 17 arabid sequence IDs for SR:

At1g09140
At1g02840
At3g49430
A14902430
At1g23860
At4g31580
At2g24590
At5g64200
At5g18810
At3g55460
At3g13570
At1g55310
At3g53500
At2g37340
At2g46610
At3g61860
At4g25500
At5g52040

These are fed into TAIR here:
https://www.arabidopsis.org/tools/bulk/sequences/

Cluster Location: /nobackup/tomato_genome/alt_splicing/SR-proteins

MaKE BLAST DB ON THE cluster:
module load blast

makeblastdb -in SRgenes-arabid.fna -input_type fasta -dbtype nucl

makeblastdb -in SRproteins.faa -input_type fasta -dbtype prot

Rerunning blast script after noticing that one of the OCR translated ID's turned a 4 into an "A". So one sequence was missing because of this.

Pulled the sequence
AT4G02430.4 | Symbols: SR34b, At-SR34b | Serine/Arginine-Rich Protein Splicing Factor 34b | chr4:1069186-1071366 FORWARD LENGTH=292

Now there are 18 Arabid sequences to explore, instead of 17.

Blastn produces only 4 hits total. Ignoring this for now as protein to protein blast searches were more abundant.

Blast results are on HPC cluster at this location:
/nobackup/tomato_genome/alt_splicing/SR-proteins

Results are these files (tab delimtied):
rw-rw--- 1 rreid2 tomato_genome 296 Oct 12 11:25 blastn-SR17cds-vs-SL5genes.txt
rw-rw--- 1 rreid2 tomato_genome 296 Oct 12 11:25 blastn-SL5genes-vs-SR17cds.txt
rw-rw--- 1 rreid2 tomato_genome 17K Oct 12 11:25 blastx-SR17cds-vs-SL5pep.txt
rw-rw--- 1 rreid2 tomato_genome 29K Oct 12 11:26 tblastn-SL5pep-vs-SR17cds.txt
rw-rw--- 1 rreid2 tomato_genome 17K Oct 12 11:26 blastp-SR17pep-vs-SL5pep.txt
rw-rw--- 1 rreid2 tomato_genome 32K Oct 12 11:27 blastp-SL5pep-vs-SR17pep.txt
rw-rw--- 1 rreid2 tomato_genome 32K Oct 12 11:32 blastx-SL5cds-vs-SR17pep.txt
rw-rw--- 1 rreid2 tomato_genome 17K Oct 12 11:32 tblastn-SR17pep-vs-SL5cds.txt

How many hits total via each blast?
*for f in *.txt; do wc -l $f ; done *
4 blastn-SL5genes-vs-SR17cds.txt
4 blastn-SR17cds-vs-SL5genes.txt
453 blastp-SL5pep-vs-SR17pep.txt
235 blastp-SR17pep-vs-SL5pep.txt
447 blastx-SL5cds-vs-SR17pep.txt
227 blastx-SR17cds-vs-SL5pep.txt
397 tblastn-SL5pep-vs-SR17cds.txt
240 tblastn-SR17pep-vs-SL5cds.txt
4 blastn-SL5genes-vs-SR17cds.txt
4 blastn-SR17cds-vs-SL5genes.txt
453 blastp-SL5pep-vs-SR17pep.txt
235 blastp-SR17pep-vs-SL5pep.txt
447 blastx-SL5cds-vs-SR17pep.txt
227 blastx-SR17cds-vs-SL5pep.txt
397 tblastn-SL5pep-vs-SR17cds.txt
240 tblastn-SR17pep-vs-SL5cds.txt

note:
SR17 = the 18 arabidopsis SR CDS / AA sequences
SL5 = the tomato genes from reference version SL5

BlastP Protein to Protein blast produces 447 hits total regardless of which set of sequences is the query and which is the reference.

Of these 447, Number of unique tomato genes soli IDs:
awk '

' blastp-SL5pep-vs-SR17pep.txt | sort | uniq | wc -l
114

BLAST RESULTS
Are located: /nobackup/tomato_genome/alt_splicing/SR-proteins

6 blast files:
for f in *.txt; do echo $f; done
blastn-SL5genes-vs-SR17cds.txt
blastn-SR17cds-vs-SL5genes.txt
blastp-SL5pep-vs-SR17pep.txt
blastp-SR17pep-vs-SL5pep.txt
blastx-SL5cds-vs-SR17pep.txt
blastx-SR17cds-vs-SL5pep.txt
tblastn-SL5pep-vs-SR17cds.txt
tblastn-SR17pep-vs-SL5cds.txt

Number of hits per file:
for f in *.txt; wc -l $f ; done

for f in *.txt; do wc -l $f ; done
4 blastn-SL5genes-vs-SR17cds.txt
4 blastn-SR17cds-vs-SL5genes.txt
453 blastp-SL5pep-vs-SR17pep.txt
235 blastp-SR17pep-vs-SL5pep.txt
447 blastx-SL5cds-vs-SR17pep.txt
227 blastx-SR17cds-vs-SL5pep.txt
397 tblastn-SL5pep-vs-SR17cds.txt
240 tblastn-SR17pep-vs-SL5cds.txt

Protein to Protein Blasts produce the most hits!

Number of Soly ID hits in Protein vs. Protein = 453

Could explore all 114 of these Soly IDs?

Or could explore the top hits:

Number of hits where Percent Identity is greater than 50%, 75% and 90%.

awk '

' blastp-SL5pep-vs-SR17pep.txt | sort | uniq | wc -l
40
awk '

' blastp-SL5pep-vs-SR17pep.txt | sort | uniq | wc -l
14
awk '

' blastp-SL5pep-vs-SR17pep.txt | sort | uniq | wc -l
0 !!!!!

Number of hits where Escore is less than 1e-10, etc….
awk '

' blastp-SL5pep-vs-SR17pep.txt | sort | uniq | wc -l
114
awk '

' blastp-SL5pep-vs-SR17pep.txt | sort | uniq | wc -l
36
awk '

' blastp-SL5pep-vs-SR17pep.txt | sort | uniq | wc -l
6

Would this be a good criteria for starting exploring genes of interest?

For Ann-curated Arabidopsis SR protein gene names and symbols and descriptions, download:

• http://lorainelab-quickload.scidas.org/quickload/A_thaliana_Jun_2009/TAIR10.bed.gz

To extra the gene identifiers, you can do something like:

grep "SR protein" TAIR10.bed | cut -f4 | cut -f1 -d . | sort | uniq | wc -l

which confirms there are 18 SR protein genes in Arabidopsis TAIR10 annotations.

Note:

The igbquickload.org site is invisible within the UNC Charlotte network due to networking issues.

The TAIR10.bed file contains 41 genes models from 18 genes encoding SR proteins, as identified in the "nomenclature" article mentioned above.

In this file, the following fields are relevant:

• field 4 - the gene model identier, e.g., AT1G02840.1
• field 13 - the nomenclature-approved gene symbol, e.g., At-SR34
• field 14 - Ann's easy-to-read, easy-to-search-for human-readable description of the gene, e.g, RNA-binding SR protein At-SR34 subfamily SR

Note that if there are multiple gene models (splice variants) for a given gene, they each get their own line in the "bed" file. Also, they all should have the same data in their 13th and 14th fields. Also, they should have the same AGI "locus" name (e.g., AT1G02840) but different transcript names (e.g., AT1G02840.1)

Note that some of the peptides Rob got from the TAIR (arabidopsis.org) Web site might not be in the above BED file. If so, please notify [~aloraine]. If this turns out to be a problem, we can instead use the data from "Araport11" BED file which contains (probably) more gene models for individual SR protein genes.

Please also see: https://bitbucket.org/lorainelab/arabidopsis-annotation-update/src/master/ - the git repository containing code Ann used to make BED files for IGB which included improved annotations for SR protein genes.

SR Splicing Factors file location:

https://docs.google.com/spreadsheets/d/1ScfcUkmf74G6eOoGGDV-3ZpQ0iqbOiVqF78SyEiPSoM/edit?usp=sharing