Cellular differentiation occurs through gene expression and protein production regulated by RNA processing and translation mechanisms. There are two major ways that differential RNA processing regulates development: 1) Through splicing, where one gene can create different proteins from alternative combinations of exons. 2) Through "censoring" or selecting different nuclear transcripts to be processed into cytoplasmic mRNA in different cell types. Translational regulation also controls developmental processes by mechanisms like phosphorylation of initiation factors that turn translation on and off.

1. Mechanism of Cellular differentiation: RNA
processing, translational regulation of
developmental process.

2. What is cellular differentiation?
• Cell differentiation is the process by which cells become specialized in order to perform
different functions.
• Differentiation occurs numerous times during the development of a multicellular
organism as it changes from a simple zygote to a complex system of tissues and cell types
• Cell differentiation is how generic embryonic cells become specialized cells.
• This occurs through a process called gene expression.
• Gene expression is the specific combination of genes that are turned on or off
(expressed or repressed), and this is what dictates how a cell functions.

3. Mechanism of cellular differentiation:
When a cell differentiates (i.e., becomes more specialized), it may
undertake major changes in its
o size,
o shape,
o metabolic activity,
o and overall function.
Because all cells in the body, beginning with the fertilized egg, contain the
same DNA, how do the different cell types come to be so different?

4.  All cells contain the same full complement of DNA,
 but each type of cell only “reads” the portions of DNA that are relevant
to its own functioning.
 In other terms, each cell has the genome but will only express specific
genes, thereby having unique proteomes.
 In biology, this is referred to as the unique genetic expression of each cell.
 In order for a cell to differentiate into its specialized form and function, it
need only manipulate those genes (and thus those proteins) that will be
expressed, and not those that will remain silent.

5. There are two major ways in which differential
RNA processing can regulate development.

6. “Splicing”
• The second mode of differential RNA processing is the splicing of the
mRNA precursors into messages for different proteins by using
different combinations of potential exons.
• If an mRNA precursor had five potential exons,
• one cell might use exons 1, 2, 4, and 5;
• a different cell might utilize exons 1, 2, and 3;
• and yet another cell type might use yet another combination .
• Thus, one gene can create a family of related proteins.

7. “Censoring”
(RNA Selection)
• The first involves the “censoring” of which nuclear transcripts are processed into
cytoplasmic messages.
• Here, different cells can select different nuclear transcripts to be processed and
sent to the cytoplasm as messenger RNA.
• The same pool of nuclear transcripts can thereby give rise to different
populations of cytoplasmic mRNAs in different cell types

9. Control of early development by nuclear RNA
selection
• In the late 1970s, numerous investigators found that
• mRNA was not the primary transcript from the genes.
• Rather, the genes transcribed nuclear RNA (nRNA), sometimes called pre-
messenger RNA (pre-mRNA).
• This nRNA is usually many times longer than the messenger RNA because the
nuclear RNA contains introns that get spliced out during the passage from
nucleus to cytoplasm.
• Originally, investigators thought that whatever RNA was transcribed in the
nucleus was processed into cytoplasmic mRNA.

10. • Cells can differ in their ability to recognize the 5´ splice site (at the beginning of
the intron) or the 3´ splice site (at the end of the intron). Or some cells could fail
to recognize a sequence as an intron at all, retaining it within the message.
• Whether a sequence of RNA is recognized as an exon or as an intron is a crucial
step in gene regulation.
• What is an intron in one cell's nucleus may be an exon in another cell's nucleus.
• Whether a spliceosome recognizes the splice sites depends on certain factors in
the nucleus that can interact with those sites and compete or cooperate with the
proteins that direct spliceosome formation.

11. • The 5´ splice site is normally recognized by
 small nuclear RNA U1 (U1 snRNA)
splicing factor 2 (SF2; also known as alternative splicing factor). The
choice of alternative 3´ splice sites is often controlled by which splice site
can best bind a protein called U2AF.
• The deletion of certain potential exons in some cells but not in others
enables one gene to create a family of closely related proteins.

12. • For instance, alternative RNA splicing enables the α-tropomyosin
gene to encode brain, liver, skeletal muscle, smooth muscle, and
fibroblast forms of this protein.
• The nuclear RNA for α-tropomyosin contains 11 potential exons,
• but different sets of exons are used in different cells.
• Such different proteins encoded by the same gene are called splicing
isoforms of the protein.

13. RNA Processing:
“The formation of mature, fully functional RNA from primary
RNA transcripts.”
or
“The process of gene expression to form a specific protein at a specific
place by the process of Translation.”
RNA serves a multitude of functions within cells.
:

14. • These functions are primarily involved in converting the genetic
information contained in a cell's DNA into the proteins that determine
the cell's structure and function.
• All RNAs are originally transcribed from DNA by RNA polymerases,
which are specialized enzyme complexes, but most RNAs must be
further modified or processed before they can carry out their roles.
• Thus, RNA processing refers to any modification made to RNA between
its transcription and its final function in the cell.

15. To become an active protein, the RNA must be:
(1) processed into a messenger RNA by the removal of introns,
(2) translocated from the nucleus to the cytoplasm, and
(3) translated by the protein-synthesizing apparatus.
In some cases, the synthesized protein is not in its mature form and
(4) must be post translationally modified to become active.
• Regulation can occur at any of these steps during development.

16. There are three main types of RNA processing
events:
1:Trimming
2:Splicing
3:Capping
4:Tailing

17. Trimming:
• Almost all RNAs have extra sequences at one or both ends of the primary
transcripts that must be removed.
Exoribonucleases.
The removal of individual nucleotides from the ends of the RNA strand is
carried out by ribonucleases (enzymes that cut RNA), called exoribonucleases.
Endoribonucleases.
An section of RNA sequence removed in the middle of an RNA strand. The
enzymes responsible are called endoribonucleases.
• Each of these ribonucleases is targeted so that it only cleaves particular RNAs
at particular places.

18. Splicing:
• RNA splicing is the removal of introns
and joining of exons in eukaryotic mRNA.
• It alsooccurs in tRNA and rRNA.
• Splicing is accomplished with the help of
spliceosomes, which remove introns
from the genes in RNA.
• The part of the RNA that is removed is
called an intron,
• whereas the two pieces that are joined
together, or spliced, are called exons.
• the splicing enzymes recognize particular
sites within the RNA, cleaves and rejoins
the RNA at those positions.

19. Capping
A 7-methylguanosine cap is
added to the 5′ end of the
pre-mRNA
• It helps in recognition of
mRNA by the ribosome
• and its protection from
RNases

20. Tailing:
At the 3' end, a poly(A) tail of 150 or more adenine nucleotides is added.
the poly(A) tail protects the mRNA molecule from
enzymatic degradation in the cytoplasm and
aids in transcription termination,
export of the mRNA from the nucleus, and translation

22. • Ribosomal RNA synthesis and processing
• occurs in a special structure within the nucleus called the nucleolus.
• The mature rRNAs bind to ribosomal proteins within the nucleolus and
assembled ribosomes
• are then transported to the cytoplasm
• to carry out protein synthesis.

23. Translation regulation of developmental process:
• Translational regulation is a widespread means of regulating gene
expression in development and cellular processes.
• It provides possibilities for controlling the spatial development of a protein
that cannot be achieved through controlling transcription alone.
• Some of the most remarkable cases of translational regulation
of gene expression occur in the oocyte.
• The oocyte often makes and stores mRNAs that will be used only after
fertilization occurs.

24. These messages stay in a dormant state until they are actived by ionic signals that
spread through the egg during ovulation or sperm binding.
Some of these stored mRNAs are for proteins that will be needed
during cleavage,
when the embryo makes enormous amounts of
chromatin,
 cell membranes, and
cytoskeletal components
 Some of them encode cyclin proteins that regulate the timing of early cell division

25. Indeed, in many species (including sea urchins and Drosophila),
maintenance of the normal rate and pattern of early cell divisions
• does not require a nucleus;
• rather, it requires continued protein synthesis
• from stored maternal mRNAs.
Other stored messages encode proteins that determine the fates of cells and
provide positional information in the Drosophila embryo.

26. • In some instances, these messages are prevented from being translated
by the binding of some inhibitory protein.
• For instance some inhibitory protein prevents the translation
of mRNA in the oocyte until fertilization.
• At that time, protein will become critical for determining which part of
the fly will be its abdomen.

27. • In other instances, the translatability of the mRNA is regulated by the length of its
poly(A) tail.
• In the Drosophila oocyte, the message remains untranslated
• until signals at fertilization allow the Cortex proteins to add poly(A) residues to
the mRNA.
• At that point, the message becomes translatable
• and its product determines which part of the embryo becomes
 the head and
 thorax

28. • Other organisms use ingenious ways of regulating the translatability of
their messages.
• The oocyte of the tobacco moth makes some of its mRNAs without their
methylated 5´ caps.
• In this state, they cannot be efficiently translated.
• However, at fertilization, a methyltransferase completes the formation of
the caps,
• and these mRNAs can be translated

29. Mechanism of Regulation of translation
in order for translation to begin,
• a protein called eukaryotic initiation factor-2 (eIF-2)
• must bind to ribosome the small subunit.
• Binding of eIF-2 is controlled by phosphorylation, or addition
of a phosphate group to the protein.

30. When eIF-2 is phosphorylated,
• it's turned "off“
• —it undergoes a shape change
• and can no longer play its role in initiation,
• so translation cannot begin.
When eIF-2 is not phosphorylated,
• in contrast, it's "on" and
• can carry out its role in initiation,
• allowing translation to proceed.

32. • In this way, phosphorylation of eIF-2 acts as a switch,
• turning translation on or off.
Inactivation of translation can be a good strategy in periods
when the cell can't “afford” to make new proteins
(e.g., when the cell is starved for nutrients

33. Translational control by eIF4E-binding proteins.
• . In cap-dependent translation, bindingof the mRNA to the small
ribosomal subunit requires the association of translation initiation
factor 4F (eIF4F) with the 5ʹ cap structure on the mRNA.
• Translational inhibition by not binding of eIF4E to the cap by an
alternative cap-binding protein IF4E2 that cannot bind eIF4.

34. • Deadenylases such as CCR4 (hemokine receptor type 4) reduce
translational activity,
• whereas poly(A) polymerases (PAPs) such as GLD2 (GLD-2 (which
stands for Germ Line Development 2) increase translational activity
by successive
• to the 3' end of specific RNAs, forming a poly(A) tail,