IN-SILICO CHARACTERIZATION OF MERISTEM DEFECTIVE (MDF) PROTEIN ASSOCIATED WITH REGULATION OF ROOT MERISTEMS IN ARABIDOPSIS

: Meristem defective protein (MDF) plays its role in proper development of meristem patterns in Arabidopsis through regulation of auxins homeostasis. It also enables plants to survive in stress. Present study was designed to characterize MDF protein in A. thaliana to get an insight into its molecular aspects. Sequence of protein was retrieved from uniprot database and subjected to CELLO, SOPMA, SWISSMODEL, MEME server and SRING tools. The protein was also docked with four proteins i. e. SR4, RSZ33, PLT1, 2 and 3. The protein was found to localize in nucleus with secondary structure comprising of alpha helix (47.20%), extended strand (6.34%), beta turn (5.37%) and random coil (41.10%). MDF was analyzed as a conserved protein with monomeric structure. Proteins observed as interacting partners of MDF included STA1, T13D8.9, LSM5, LSM2, LSM4 and LSM8. Docking analysis revealed highest and lowest affinities of MDF binding with RS4, RZS33, PLT1, PLT2 and PLT3 in case of FGRTLTPKEAFRLLSHKFHG and IQGQTTHTFEDLNSSAKVSSDYFSQ conserved motifs, respectively. Features of MDF protein explored in this investigation can be exploited for engineering and production of plants with efficient root meristems development.


INTRODUCTION
Meristem defective protein (MDF) also known as Defectively Organized Tributaries 2 (DOT2) is encoded by a splicing factor gene.MDF is a serinearginine (SR) related arginine-serine (RS) protein comprising of a polypeptide of 820 amino acids (Kakkar, 2019).Sequence of this protein is homologous to squamous cell carcinoma antigen recognized by T-cells 1 (SART1) human protein (de Luxán-Hernández et al., 2023).MDF has two motifs i. e. nuclear localization Cterminus and RNA-binding N-terminus.This protein regulates the development of root meristems and cell division (Kakkar, 2019).
Arabidopsis thaliana commonly called as thale cress and mouse-ear cress belongs to family Brassicaceae.Complete genome annotation, shot term reproductive cycle, easy transformations, simple structure of roots, and susceptibility to artificial methods of mutation inductions, has made this plant an excellent model organism for research purpose (Fonseka et al., 2013;Fouracre and Poethig, 2019;Montazeaud et al., 2023;Pasternak et al., 2023).Several genes involving in arsenic resistance, bud and flower development, elongation of primary and lateral roots, photosynthesis and phytohormones synthesis have been deeply investigated in A. thaliana (Alvarez et al., 2023;de Luxán Hernández, 2022;Lin et al., 2023;Ranjan et al., 2023;Smyth, 2023;Zeng et al., 2023).
MDF protein, via alternative splicing, regulates the concentration as well as the distribution of auxins in roots in A. thaliana.It also plays role in inhibiting the cell death as well as differentiation in root meristems under the conditions of stress (Calixto et al., 2018).Alternative splicing induces diversity at the level of transcriptome and proteome through encoding a large number of transcripts from smaller number of genes.During this process, MDF plays crucial role in spliceosome assembly.A study involved the generation of MDF mutants in A. thaliana revealed defective meristems contributed by abnormal transcriptome variations (Casson et al., 2009;de Luxán-Hernández et al., 2023).
During spliceosome complex formation, ribonucleoproteins U1 and U2AF attach to 5' and 3' splice sites, respectively.The later ribonucleoprotein is also bound by polypyrimidine tract i. e. YYY-rich.This step follows the binding of MDF protein with exon enhancing splicer sequence (ESE) of pre-mRNA via its N-terminus end.Afterwards, the U2 and U2AF binding induce conformational change in RNA molecules which promotes binding of tri-snRNP complex of three ribonucleoproteins i. e. U1, U5 and U6 (Wang et al., 2008).Association of MDF with this tri-snRNP complex has been reported to regulate proper splicing of 2037 transcripts (de Luxán-Hernández et al., 2023).In addition to this, MDF controls splicing via interacting with other proteins like SR34, RSZ33, ACC1, PIN family of proteins and PLETHORA gene family i.e. PLT1, 2 and 3 (Figure 1) (Kakkar, 2019;Thompson et al., 2023;Wisniewska et al., 2006).

Figure 1: Role of MDF protein in spliceosome assembly and auxins homeostasis
Keeping in view the significance of MDF protein in plant roots development and the wide adaptability of A. thaliana as model system, we initiated present study to target the characterization of MDF protein in A. thaliana.This characterization included the prediction of sub-cellular localization, two dimensional (2D), three dimensional (3D) configuration, conserved domains and interactions with other proteins.This analysis can be helpful in designing strategies for optimizing activity of this protein in plants root growth under stress conditions not only in A. thaliana but also in economically important crops.

METHODOLOGY
Sequence retrieval from Uniprot database: Sequence of MDF protein with accession no.Q9LFE0 in A. thaliana was retrieved from Uniprot database (https://www.uniprot.org,accessed in Sep 2023) (Consortium, 2019)

CELLO: prediction of sub-cellular localization:
To predict the localization of MDF protein at sub cellular level, CELLO tool (cello.life.nctu.edu.tw,accessed in Sep 2023) was employed (Xiong et al., 2016).

MEME server: prediction of conserved domains:
MEME suite version 5.5.5 (Multiple Em for Motif Elicitation) was employed (https://memesuite.org/meme/tools/meme,accessed in Sep 2023) (Bailey et al., 2015).For this analysis, for motif site distribution the option of any number of repetitions (anr) and 10 number of motifs was selected.

Three dimensional (3D) structure:
The template identified was SART-1 family protein DOT2 with 100% sequence identity.The GMQE value was observed to be 0.66 (Figure 3).MDF protein was monomeric with highly complex folding (Figure 3).

Conserved domains:
The conserved domains predicted at twenty different locations of protein through MEME suite.The p-values ranged between 1.05e-8 and 8.18e-12.The E-values were recorded to be 1.0e+002 to 7.0e+001 (Table 1 and Figure 4).
Interaction with other proteins: Stabilized 1 (STA1) is a pre-mRNA splicing factor, T13D8.9 is an RNA recognition motif containing protein (RRM), Sm like proteins (LSM5, LSM2, LSM4 and LSM8) which are the components of LSM protein complex that is associated with RNA processing, U5 small nuclear ribonucleoprotein component (CLO) which is a 110kDa splicing factor involved in pre-mRNA splicing, small nuclear ribonucleoprotein SmD2 (F17A22.3),U5 small nuclear ribonucleoprotein component (GFL) which is a 109kDa 110kDa splicing factor involved in pre-mRNA splicing and thioredoxin like U5 (Q6NMD4-ARATH) which is mRNA splicing factor.The scores recorded were above 0.9 in all the cases (Figure 5).

DISCUSSION
Auxins are associated with patterning of root meristems at embryonic stages in A. thaliana.MDF protein has central role in auxins homeostasis.It regulates the spliceosome assembly and expression of various genes which are associated with auxins transport and meristems development (Shen, 2019).So far, no one has targeted the comprehensive characterization of MDF protein using in-silico tools.Therefore, present work is the first ever project reporting it's in silico analysis.
Comparison of present study findings with literature revealed that sub-cellular localization of MDF in nucleus is consistent with previously reported work.
The study analyzed location of protein using a different in silico tool i. e. Plant P-Loc protein localization software (Chou and Shen, 2007).Assessment of 3D configuration via SWISSMODEL using template search mode, revealed the similarity of MDF with human hSART-1 protein which is also consistent with previous findings (Blencowe et al., 1999).Multiple conserved domains were identified in present study which is inconsistent with literature reporting only RNA binding and nuclear localization motifs and no conserved domains in MDF (Terribilini et al., 2007).An initial study has reported the interaction of MDF with STA1 protein, hence our finding is consistent with literature.However, in the same study, no interaction was observed between MDF and LSM (Thompson et al., 2023).
To maintain the auxins homeostasis and distribution, MDF regulates the expression of several other proteins.A study conducting using MDF mutants revealed the reduced level of PLT transcripts leading to disruption of root meristem patterning (Casson et al., 2009).Another gene RSZ33 encodes alternatively spliced mRNA which is abundantly present in root meristems.In a project, mis-spliced RSZ33 transcripts were reported in MDF mutants causing retarded root meristems (Thompson et al., 2023).Keeping in view these interacting genes of MDF, three long conserved domains predicted in the analysis of MDF protein, were selected for docking against SR4, RSZ33, PLT1, PLT2 and PLT3 proteins.This helped to check out the binding tendency of MDF with these protein ligands.It was found that the motifs in SR4, RSZ33, PLT1, PLT2 and PLT3 proteins, respectively have higher binding tendencies with MDF.These domains can be targeted for modifying MDF regulatory activity of spliceosome, thus improving its function of meristems development.Literature does not report any docking analysis of MDF protein of A. thaliana.

Conclusion:
Analysis at the molecular level of MDF and its interactions predicted in present study might be helpful to explore the molecular mechanisms underlying its activity.Through modifications in MDF protein the plants seeds responses to stressors should be evaluated in different important plants.The protein imparting highest stress tolerance can be useful in development of good quality crops.

Statements and Declarations
Funding: N/A

Figure 2 :
Figure 2: In-silico prediction of sub-cellular localization and 2D configuration of MDF protein (a) CELLO tool (b) SOPMA tool

Figure 4 :
Figure 4: Prediction of conserved domains of MDF protein based on MEME suite.

server: Docking of MDF conserved motifs with ligands: Docking
PLT1, PLT2 and PLT3.For analysis, HDOCK server (hdock.phys.hust.edu.cn,accessed in Sep 2023) was employed and docking score, confidence score and RMSD values were determined