My Masters seminar on Structural biology of Nucleocytoplsmic transport @UAS Raichur

1. 1

2. STRUCTURAL BIOLOGY OF
NUCLEOCYTOPLASMIC TRANSPORT
MANOJ N.S
PG19AGR11055
Dept of Genetics and plant breeding
UAS , RAICHUR
Date : 29/05/2020
SEMINAR-1
On

3. Flow of seminar
Introduction
Nuclear pore complex
Structure of nuclear pore complex
Mechanism of import and export of protiens
Rangtpase cycle
Rna transport
Research evidences
conclusion

4. What is NucleoCytoplasmic Transport ?
• Transport of thousands of macromolecules between
cytoplasm and nucleus occurs through nuclear pore
complex (NPC).
• Nucleocytoplasmic transport is regulated by complex
interplay of nuclear transport machinery which includes
elegant structure like nuclear pore complex ,nuclear
envelope protiens and transport receptors.

5. Why NC transportation is needed ?
• Nuclear envelope separates nucleoplasm from cytoplasm and controls
the nucleocytoplamic transport which is critical for many physiological
and pathological cellular process including gene expression,signal
transduction and oncogenesis.
• RNA transcripts are exported from the nucleus to reach the ribosomes
in the cytoplasm. Proteins such as histones, polymerases, transcription
factors, are imported into nucleus to reach the genetic material.
• Site of DNA replication and RNA biogenesis takes place in the nucleus.
• Site of Protein synthesis is cytoplasm.

6. NUCLEAR ENVELOPE
• Nuclear membrane is a physical barrier b/w nucleoplasm and cytoplasm.It
is a phospholipid bilayer which consists of inner and outer membrane and
are separated by perinuclear space.
• Outer membrane is in continuity with rough endoplasmic reticulum
studded with ribosomes which are engaged in protien synthesis.
• Nuclear membrane is perforated by NPC which fuse the outer nuclear
membrane and inner nuclear membrane together and creates a channel
for nucleocytoplasmic transport.

7. Nuclear Pore Complex
NPC is a multiprotein complex that serve as key regulator of molecular
trafficking b/w cytoplasm and nucleus.

8. STRUCTURE OF NUCLEAR PORE
COMPLEX
SOROKIN et al.

9. Nuclear pore complex
Http7:Annual review in biochemistry/nuclear pore complex

10. CHARACTERISTICS OF NUCLEOCYTOPLASMIC
TRANSPORT
Selectivity:
 RNA doesn’t goes through unless accompanied by specific protiens that
act as carriers ,DNA doesnt go through it.
Regulation Of Signals :
 Protiens going into the nucleus must have a nuclear localization signal (
NLS) usually a sequence of 4 or more closely located positively charged
amino acids.
 Protiens going out of nucleus must have a nuclear export sequence usually
a series of leucine with characteristic spacing b/w them.

11. TERMINOLOGIES
 TRANSPORT RECEPTORS
 CARGO
 KARYOPHERINS (CARRIER PROTIENS)

12. Mechanism of import and export
Importin + Cargo ( In cytoplasm )→
releases in nucleus on binding with
RanGT
Exportin + Cargo + RanGTP (In
nucleus) → releases on conversion of
RanGTP into RanGDP
Atlanta cook et al

13. IMPORT AND EXPORT OF CARGO
Http7:Annual review in biochemistry/import and export of cargo

14. Ran gtpase cycle
• Ran exists in two conformations depending upon binding of GTP and GDP
• Ran GTPase is very high concentration in the nucleus whereas RanGDP is
very high concentration in cytoplasm.
• Conversion between GTP and GDP is triggered by two Ran specific
regulatory protiens that includes a cytosolic gtpase activating protien GAP
and a nuclear guanine exchange factor GAF.

15. Atlanta cook et al

16. How Directionality Achieved ?
• RanGDP / RanGTP Con.
Gradient
• RanGTP gradient is
maintained by asymmetric
distribution of GAP & GEF
• RanGTP stabilizes export
complex
• RanGDP stabilizes import
complex
SARA NAKIELNY et al

17. Transport Receptor – NTF2
• FG-Nup binding to NTF2
at the spot where Ran
binds upon import
• NTF2 vital for proper
balance of components
across Nuclear envelope
• Nuclear import of
RanGDP is mediated by
NTF2.
• NTF2 spec. recognized by
RanGDP
TIMOTHY et al

18. RNA TRANSPORT
 The RNA molecules undergo a variety of post transcriptional processing
events, after which they are transported to their sites of function
throughout the cell.
 the subcellular locations of each type of RNA namely messenger RNA
(mRNA), small nuclear RNA (snRNA), ribosomal RNA (rRNA), and transfer
RNA (tRNA) are critical to the normal functioning of the cell.
 majority of RNAs are transported to the cytoplasm. Some RNAs need to be
reimported to the nucleus for their function.
 All cellular RNA species exit the nucleus through NPCs by an active,
mediated mechanism

19. TRANSPORT OF mRNA
Processing events that influence Nuclear Export
Transcribed pre-mRNAs undergo three co/post transcriptional processing
events.
1. 5’ capping
2. Splicing
3. polyadenylation

20. MECHANISM OF m RNA TRANSPORT
• Trancribed in the nucleus by DNA polymerase II
• Majority of m RNA export is independent of Ran & karyopherin β family.
• The (TAP:p15) m RNA export factor is a heterodimer which helps in Mrna
transport.

21. TRANSPORT OF tRNA
• Transcribed in the nucleus by RNA polymerase Ш.
• t RNA molecules have to be processed before transport into th cytoplam.
• Further modification of tRNA occurs in the cytoplasm.
• Processing comprises
1. Excision of 5’ & 3’ terminal sequences.
2. Addition of CCA to the 3’ end.
3. Base modification.
4. Removal of introns and base editing.

22. RIBOSOMAL RNA TRANSPORT
• Transcribed in the nucleus by RNA polymerase I
• Nuclear export occurs in context to Ribosomal-subunits
• Transport requires energy & through NPC

23. THEMES & PERSPECTIVES
• Common theme :
transport in both
direction across
the NPC mediated
by RNA-binding
proteins

24. FUTURE ISSUES
• In order to determine the general principles of Ran-dependent transport,
the structure of each karyopherin bound cargoes is required.This will
reveal the mechanisms by which nuclear import/export signals,
particularly of cargoes such as NESs and NLS are recognized.
• In some of the cases, the same β-karyopherin can mediate both import
and export so there is need to know extensive studies on karyopherin.
• mRNA export pathway which is one of the complex process and different
mRNAs are recognized for export, process through which it is released to
the cytoplasm hence the nuclear transport machinery performs is need to
be known clearly.

25. Nuclear RNA Export and Its Importance
in Abiotic Stress Responses of Plants
V. Chinnusamy , Z. Gong , and J.-K. Zhu
The transduction of abiotic stresses to switch on genes involved in adaptive responses
are critical to the survival and reproduction of plants exposed to adverse environments.
 Plants have evolved multiple stress response pathways, some of which are specific, but
others may be common for various abiotic stresses
Nucleocytoplasmic trafficking—entry of developmental/environmental signals
for gene expression and export of expressed gene products from the nucleus
through nuclear pores—plays a fundamental role in gene expression in eukaryotic
organisms.

29. Role of mRNA Export in Abiotic Stress Response of Plants
• Dead box proteins (DBPs) with RNA helicase activity are involved in RNA metabolism
such as transcription, RNA processing, RNA decay, and nucleocytoplasmic transport. In
yeast and vertebrates, Dbp5 plays a crucial role in export competent Mrnp formation
in nucleus, transport of mRNP through the NPC, and release of mRNA from mRNP in
the cytosol. The role of DBP in mRNA export and abiotic stress response of flowering
plants came from the analysis of the mutant los4 (low expression of osmotically
responsive genes 4) of Arabidopsis .
(GONG et al)
• Role of nups in abiotic stres reaponae was demonstrated ATPNUP 160 mutant which
was hypersensitive to chilling stress .Map based cloning of ATNUP 160 revealed it to
NUP homologus to mammal NUP 160 ATNUP 160 flower show early flowering ATNUP
160 is critical for mrna export , cold resposnive genes expression ,cold tolerance as
well as plant development at normal temperatures
( Dong et al 2006 . Perry et al )

30.  SAD 2 domain enhanced gene expression and stress response genes under
cold ABA hypersensitivity in seed germination and seedling growth , SAD 2 is
localized in the nucleus thus result suggested that sad 2 play a crucial role in
nuclear regulator of cold .
(versules et al .2006)
 The cold-sensitive phenotype of los4–1 showed inhibited mRNA export
under cold stress conditions. In contrast, the cold-tolerant but heat-sensitive
los4-2 showed normal mRNA export under cold stress but was defective in
mRNA export from the nucleus at warm temperatures (Gong et al. 2005). The
results from los4 mutants demonstrated that LOS4 DEAD-box RNA helicase
plays an important role in mRNA export, which is necessary for abiotic stress
response.
(Gong et al. 2005).

31. CONCLUSION
NUCLEOPHORIN (NUP107–160 complex) proteins shown that they participate
in mRNA export and regulate germination,flowering,hormone response,host-
plant symbiotic interaction, and biotic and abiotic stress tolerance. Further
studies on various components of the nuclear mRNA export machinery in
plants and their role in abiotic stress response will be necessary to better
understanding the link between mRNA export and abiotic stress-responsive
gene expression.

33. EgRBP42 from oil palm enhances adaptation to stress in
Arabidopsis through regulation of nucleocytoplasmic transport of
stress-responsive mRNAs
Wan-Chin Yeap1*, Parameswari Namasivayam2, Tony Eng Keong Ooi1, David Ross Appleton1,
Harikrishna Kulaveerasingam3 and Chai-Ling Ho2, 4*
( Sime Darby Plantation Berhad, Research and Development, Biotechnology and Breeding, Sime
Darby Technology Centre Sdn. Bhd., Serdang, Selangor Darul Ehsan, 43400 Malaysia)
OBJECTIVE - , an hnRNP-like RNA-binding protein from oil palm, which
could be necessary for rapid protein translation to confer abiotic stress
tolerance in plants.

34. MATERIALS AND METHODS
• Six-month-old oil palm seedlings (Dura) were used for abiotic stress
treatments and gene expression analysis.
• Wildtype Arabidopsis, transgenic lines of WT:Pro35SEgRBP42 and mutant
atuba2c were grown under controlled environment conditions at 22 °C, 65
% relative humidity and 120 μmolm-2 s-1 illumination under long day
conditions, 16 h of light followed by 8 h of darkness.
• Pro35S:EgRBP42 in pEG100 and pEG100 (wildtype) were coated on the
surface of gold particles and bombarded into leaf tissue sections, Biolistic
Particle Delivery System bombardments were conducted .
• Total RNA was extracted from oil palm leaves, Arabidopsis leaves and
Arabidopsis flower using NucleoSpin RNA plant (Macherey-Nagel, Düren,
Germany)and observed target gene expression between wildtype .

36. Overexpression of EgRBP42 confers abiotic stress tolerance and accelerates
recovery in vegetative development

37. Constitutive overexpression of EgRBP42 accelerates flowering in Arabidopsis
under normal and stress conditions

39. EgRBP42 binds to the AG-rich motifs in the coding regions of target transcripts.

40. EgRBP42 interacts with proteins localized in the nucleus and the cytoplasm

42. EgRBP42 protein accumulation and localization is mediated by abiotic stress.

44. Post-transcriptional regulatory model for EgRBP42-mediated RNA metabolism in
response to abiotic stress in oil palm.

45. CONCLUSION
 The overexpression of EgRBP42 in Arabidopsis confers
drought, cold, salinity and flood tolerance with enhanced
post-stress recovery. Through this we can propose a model
describing post-transcriptional nucleocytoplasmic transport of
stress-responsive mRNAs in response to different abiotic
stresses, leading to a better adaptation to environmental
changes