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Background

Protein of interest: SRp20

Significance: RNA binding protein

Organism: Homo Sapiens, Streptococcus sp. Group G

Characteristics: Ser/Arg-rich domain

Basic function: Splicing Factor

164 aa: half of it belongs to the RS (Arg/Ser) domain

MW: 19 kDA

Determined via HSQC NMR (heteronuclear single quantum coherence)

Function

General Funtions (GO INTO DETAIL ABOUT EACH)

RNA processing

Termination of transcription

Protein translation

Cellular proliferation/maturation

Involved in Insulin signaling (loss in liver results in wrong splicing of glucose metabolism genes)

Embryogenesis

Splicing Factor

The spliceosome is a complex responsible for splicing pre-mRNA to mature mRNA, composed of snRNA and associated proteins

Introns are spliced out, while exons are ligated together

Splicing is necessary for mRNA maturation, enabling it to exit the nucleus

Alternative Splicing

Alternative splicing allows many different proteins to be produced from one mRNA by splicing different combinations of introns and exons

Changes or defects in this process linked to human diseases

RNA processing factors may be targets for future therapies

SR Proteins

Characteristics:

One or two N’ term RRMs (RNA recognition motifs)

Arg/ser-rich domain downstream

Most serine residues phosphorylated

The N’ term RRM provides substrate specificity

The C’ term RS domain aids protein-protein interactions (simplifying spliceosome recruitment)

RMM

N-terminal RNA recognition motif

standard for an RRM

A four stranded β-sheet and two α-helices

Most commonly, three aromatic side-chains (in β-3 and β-1 strands) accommodate two nucleotides

Recognition enables binding of SRp20 to RNA

RRM Motif

mRNA Maturation

Relationship to other proteins

9G8

RRM Motif in SRp20

RRM Motif in 9G8

SRp20 and 9G8 Sequence Alignment

Disease

Cancer

Oncogenic signaling pathways such as the WnT pathway cause-effect relationship with increase expression of SRp20

Downregulation of SRp20 promotes cellular senescence through AS of TP53 and generating p53 (tumor suppressor gene)

Silencing SRp20 in cancer cells: slower cell proliferation

Activator of the AS of gene CD44

Pyruvate Kinase M (PK-M gene)

Regulate Rac1b protein expression in colorectal tumor cells

Alzheimer's

TRKB: AS results in TrkB-Shc transcript

Human Papillomavirus: HPV

Increases SRp20 which in turn regulates the gene expression of HPV via interaction with A/C-rich RNA nucleobases Overexpression of it involved in HPV-induced cancers such as cervical cancer

Structural highlights

Poor Solubility Problem

The SRp20 protein has poor solubility in its free state. This made it impossible to determine the structure of SRp20 using HSQC Spectroscopy without a modification to the free state protein. This problem was resolved by studying the proteins after fusing the RRM (RNA-recognition motif) with the immunoglobulin G-binding domain 1 of Streptococcal Protein G GB1 solubility tag.

SRp20 with solubility tag

RNA Interactions

1H-15N HSQC results showed a large hydrophobic β-sheet on the RRM binds to the RNA with all four bases contacting one of the four aromatic residues” (hydrophobic interactions) Other structural studies show that amino acids of the β-hairpin are directly hydrogen bonded to bases of nucleic acid targets Using a smaller peptide chain reduced the NMR broadening seen with longer peptides (allowing for structure determination), though the binding affinity was also reduced

C1 and A2 stack on Y13 in β1 and F50 in β3 (aromatic side chains), respectively.

The residue F48 inserts between the sugar rings of C1 and A2

Closer look at the ligand used

Looking at the ligand

The conformation of U3 and C4 is unusual because U3 bulges out while C4 stacks over A2, partially.

C4 and A2 interactions with the ligand

also adopts an unusual syn conformation

A2 adopts an unusual syn conformation

C4 is maintained in its position by a hydrogen bond between C4 amino group and the A2 2’ oxygen (IMAGE)

C4 is maintained in its position by a hydrogen bond between C4 amino group and the A2 2’ oxygen

RRM Domain Interactions

U3 interacts with Phe 48, Trp 40, Ala 42, and with the β2-3 loop of the RRM (IMAGE)

U3 interacts with Phe 48, Trp 40, Ala 42, and with the β2-3 loop of the RRM

C1 Binding Pocket

C1 amino protons hydrogen bond with Leu 80 carbonyl oxygen and Glu 79 side-chain carbonyl oxygen. C1 N3 hydrogen bonds with Asn 82 amide. C1 O2 hydrogen bonds with Ser 81 hydroxyl group.

Ser-Arg Rich Domain

Specificity

4 nt can be accommodated by RRM β-sheet, but recognition is only partially sequence specific. CAUC C1 more specific, A2 and U3 less specific It is uncertain whether C4 is specifically recognized by the RRM A is prefered over G at the 2 position, but no indication of preference over U or C U3 is even less specific, could be C, G or A The recognition of C1 is functionally necessary because a C to G mutation within the histone mRNA can impair RNA export

Advantages of low specificity

Less evolutionary pressure on bound RNA (ideal for exonic sequences) More RNA sequences can be targeted SRp20 can associate/disassociate with RNA more easily Important for highly dynamic RNA metabolism processes RNA binding affinity can be modulated by protein-protein interactions (which are dependant on the level of phosphorylation) This can be used to tune post-transcriptional gene expression

Relevance and Conclusions

Understanding and recognizing the mechanisms that SRp20 is involved in can help find treatment and management of cancer patients The use of SR proteins (such as SRp20) may in the future be used for targeted therapy No structure for the C-term domain

Future Directions

Recent structural studies emphasize that not only the β-sheet surface but also the loops connecting β-strands and a-helices can be crucial for nucleic acid recognition → future research looking more closely at this possibility Farther research could be done investigating the process of RS domain phosphorylation and how it controls splicing Look farther into the specificity or lack of specificity relating to SR proteins Mutate specific residues in order to map out SRp20 mechanistic function

References

Cle’ry A, Blatter M, Allain FHT. 2008. RNA recognition motifs: boring? Not quite. Current Opinion in Structural Biology 18: 290–298. Doi: 10.1016/j.sbi.2008.04.002

Corbo C, Orrù S, Salvatore F. 2013. SRp20: An overview of its role in human diseases. Biochemical and Biophysical Research Communications 436: 1–5. Doi: 10.1016/j.bbrc.2013.05.027

Hargous Y, Hautbergue GM, Tintaru AM, Skrisovska L, Golovanov AP, et. al. 2006. The EMBO Journal ) 25: 5126–5137. Doi: :10.1038/ sj.emboj.7601385