Write brief notes on a) RNA transport b) Processing of tRNA?

Ans) RNAtransport: RNA molecules synthesized in the nucleus are transported to their sites of function
throughout the eukaryotic cell by specific transport pathways. This review focuses on transport of messenger
RNA, small nuclear RNA, ribosomal RNA, and transfer RNA between the nucleus and the cytoplasm.

What Happens During RNA Transport?

Combining RNA transport with control of translation is a key way to direct protein production to specific parts of a
cell or organism. mRNAs link up with proteins that control every step of their life cycle while they are being moved.
Together, mRNAs and proteins make up large ribonucleoprotein (RNP) complexes. In these complexes, different
factors control how localised mRNAs are put together, kept stable, translated and moved. The complexes are then
taken to their final destination by microtubules, microfilaments, and their motors. So that proteins can be made at
the final target site, the translation of mRNAs that are being moved must be turned off during the trip and turned
back on only when the mRNAs arrive at their final destination.
Even though several proteins that control the translation of localised transcripts have been identified, only a few
mRNAs have had their translation slowed down during transport and their local protein synthesis turned on at their
final destination. In this paper, we will focus on some of the best-studied examples of how localised transcripts are
controlled by translational regulation. We will also look at whether the complexes that control both localization and
translation are found in other eukaryotes. We will also try to find out more about how eukaryotes keep the
connection between where mRNA is and how it is translated.

How is RNA Transported out of the Nucleus?

The process of nuclear export is similar to doing the opposite of nuclear import. During nuclear export, the exportin
binds the cargo and Ran-GTP in the nucleus before diffusing through the pore into the cytoplasm. In the cytoplasm,
the complex then dissociates. The resultant Ran-GDP complex is returned to the nucleus, where it performs the
process of exchanging the binding ligand for GTP. Ran-GTP is then able to connect to GAP and hydrolyze GTP.
Therefore, whereas importins depend on RanGTP to dissociate from their cargo, exportins require RanGTP in order
to bond to their cargo. This is because exportins bind RanGTP to their cargo.
After the post-transcriptional modification process is finished, mature mRNA is transported to the cytoplasm by a
protein that is specifically designed to act as an mRNA exporter. Although the particular mechanism behind this
translocation process is not yet fully understood, this process is actively dependent on the Ran protein. Some genes
that are transcribed more frequently than others are physically situated close to nuclear pores in order to make the
process of translocation easier.
The export of tRNA is likewise dependent on the numerous alterations that it goes through; as a result, the export of
tRNA that does not function properly is prevented. This quality control system is essential because of the crucial
role that tRNA plays in the translation process. During this process, tRNA is responsible for adding amino acids to a
peptide chain that is expanding. Exportin-t is the name given to the tRNA exporter found in vertebrates. The
presence of RanGTP helps to speed up the process by which exportin-t connects directly to its tRNA payload that is
located in the nucleus. tRNA’s capacity to attach to exportin-t and, as a consequence, to be exported is inhibited
when mutations that change the structure of tRNA occur. This provides the cell with an additional quality control
step.

Conclusion
In order to support the active metabolism of distant dendritic and axonal compartments, neurons decentralise
protein synthesis away from the cell body. RNA trafficking over long distances is facilitated by the neuronal RNA
transport machinery, which is composed of cis-acting RNA regulatory elements, transport granule proteins and
motor adaptor complexes. An increasing number of neurodegenerative illnesses have been linked to dysfunctional
RNA transport, which has been demonstrated to be a common pathomechanism in recent years by improvements
in human genetics, subcellular biochemistry and high-resolution imaging. RNA transport is dissected in this review
to examine the role played by each component in RNA localization and the specific contributions made to
neurodegeneration by each one of these components.
b) Processing of tRNA
The processing of mature tRNA is done from these precursor transcripts by
nucleases that cleave and trim them with high precision. In prokaryotes, this
process involved deletion of 5' leader sequence, eradication of 3' terminal non
essential residues and chemical modifications of assorted bases and ribose
units.
ransfer RNA (tRNA)

Each different tRNA binds to a specific amino acid and transfers it to the ribosome. Mature tRNAs
take on a three-dimensional structure through intramolecular basepairing to position the amino acid
binding site at one end and the anticodon in an unbasepaired loop of nucleotides at the other end.
The anticodon is a three-nucleotide sequence, unique to each different tRNA, that interacts with a
messenger RNA (mRNA) codon through complementary base pairing.

There are different tRNAs for the 21 different amino acids. Most amino acids can be carried by more
than one tRNA.

Figure: Structure of tRNA: This is a space-filling model of a tRNA

molecule that adds the amino acid phenylalanine to a growing polypeptide chain. The anticodon AAG binds the codon
UUC on the mRNA. The amino acid phenylalanine is attached to the other end of the tRNA.

In all organisms, tRNAs are transcribed in a pre-tRNA form that requires multiple processing steps
before the mature tRNA is ready for use in translation. In bacteria, multiple tRNAs are often
transcribed as a single RNA. The first step in their processing is the digestion of the RNA to release
individual pre-tRNAs. In archaea and eukaryotes, each pre-tRNA is transcribed as a separate
transcript.

The processing to convert the pre-tRNA to a mature tRNA involves five steps.

1. The 5′ end of the pre-tRNA, called the 5′ leader sequence, is cleaved off.

2. The 3′ end of the pre-tRNA is cleaved off.

3. In all eukaryote pre-tRNAs, but in only some bacterial and archaeal pre-tRNAs, a CCA sequence of
nucleotides is added to the 3′ end of the pre-tRNA after the original 3′ end is trimmed off. Some
bacteria and archaea pre-tRNAs already have the CCA encoded in their transcript immediately
upstream of the 3′ cleavage site, so they don’t need to add one. The CCA at the 3′ end of the mature
tRNA will be the site at which the tRNA’s amino acid will be added.

4. Multiple nucleotides in the pre-tRNA are chemically modified, altering their nitorgen bases. On
average about 12 nucleotides are modified per tRNA. The most common modifications are the
conversion of adenine (A) to pseudouridine (ψ), the conversion of adenine to inosine (I), and the
conversion of uridine to dihydrouridine (D). But over 100 other modifications can occur.

5. A significant number of eukaryotic and archaeal pre-tRNAs have introns that have to be spliced
out. Introns are rarer in bacterial pre-tRNAs, but do occur occasionally and are spliced out.

After processing, the mature pre-tRNA is ready to have its cognate amino acid attached. The cognate
amino acid for a tRNA is the one specified by its anticodon. Attaching this amino acid is called
charging the tRNA. In eukaryotes, the mature tRNA is generated in the nucleus, and then exported
to the cytoplasm for charging.

2) Write a detailed note on different mechanisms of translation regulation in eukaryotes?

Regulation of Translation in Eukaryotes

May 25, 2023 by Sagar Aryal

Edited By: Sagar Aryal

Translational regulation refers to the control of the levels of protein synthesized from
its mRNA.
In eukaryotes, regulation of protein synthesis can occur by modification of DNA or at the
level of transcription within the nucleus, processing of mRNA in the nucleus, or translation
in the cytoplasm.
A. Regulation through Changes in Genes
1. Genes can be lost (or partially lost) from cells so that functional proteins can no
longer be produced (e.g., during differentiation of red blood cells).
2. Genes can be amplified. For example, the drug methotrexate causes hundreds of
copies of the gene for the enzyme dihydrofolate reductase to be produced, which
results in resistance to the drug.
3. Segments of DNA can move from one location to another on the genome,
associating with each other in various ways so that different proteins are
produced. Example: A number of different potential sequences (or arrangements)
occur for various portions of an antibody-producing gene.
4. Modification of the bases in DNA affects the transcriptional activity of a gene.
• Cytosine can be methylated at its 5 position, which often occurs in CpG
islands within promoter regions.
• The greater the extent of methylation, the less readily a gene is transcribed.
B. Regulation of the level of transcription
1. Histones, which are small, basic proteins associated with the DNA of eukaryotes,
act as non-specific repressors.
• Histone acetyltransferases, or acetylases (HAT or HAC) will acetylate lysine side
chains on histones, which reduces the charge attraction between histones and
DNA.
• Histone deacetylases (HDAC) will remove the acetate groups from histones,
thereby allowing histones to reassociate with the DNA.
• Heterochromatin is the tight association of histones and DNA, and represents
the transcriptionally inactive areas of the genome.
• Euchromatin refers to the transcriptionally active areas of the genome in
which histone association with the DNA has been reduced.
2. The expression of specific genes is stimulated by positive mechanisms.
3. Inducers (e.g., steroid hormones) enter cells, bind to protein receptors, interact
with chromatin in the nucleus, and activate specific genes.
4. Some genes have more than one promoter. Thus, the promoter that is used can
differ under varying physiologic conditions or in different cell types.
C. Regulation by Chromatin Remodeling
Nucleosome displacement such that transcription can occur
1. An ATP-driven chromatin remodeling complex will bind to the regions of DNA that
contain acetylated histones. Bromodomains on proteins within the complex
recognize the acetylated histones. Once bound, using ATP as an energy source, the
complex will move and displace histones to free up an area of DNA for
transcription.
2. Histone acetylase activity is often associated with transcription factors that bind to
the region of DNA that needs to be transcribed, facilitating the removal of
histones from the DNA and binding of the transcription apparatus.
D. Regulation during processing and transport of mRNA
Regulatory mechanisms that occur during capping, polyadenylation, and splicing can alter
the amino acid sequence or the quantity of the protein produced from the mRNA.
Editing of mRNA also occurs, and the rate of degradation of mRNA is also regulated.
1. Alternative splice sites can be used to produce different mRNAs.
2. Alternative polyadenylation sites can be used to generate different mRNAs.
3. mRNAs can be degraded by nucleases after their synthesis in the nucleus and
before their translation in the cytoplasm.
4. RNA editing involves the alteration (“editing”) of bases in mRNA after
transcription.
5. Small and interfering RNA (SiRNA)
• Gene silencing can occur through the use of small RNA products (miRNA),
which can either block the translation of a target mRNA or induce
degradation of the target mRNA.
 • miRNA molecules are the products of many genes scattered throughout the
chromosome, some even located in the introns of the genes they regulate.
miRNAs are synthesized in the nucleus and processed to form an active
molecule that will bind to the target RNA and ablate its expression.
E. Regulation at the translational level
It occurs during the initiation or elongation reactions.
1. Heme stimulates the synthesis of globin by preventing the phosphorylation and
consequent inactivation of eIF-2, a factor involved in the initiation of protein
synthesis.
2. Interferon stimulates the phosphorylation of eIF-2, causing inhibition of initiation.
3. Iron-response elements (IREs) in mRNA for ferritin (an iron storage protein) and
the transferrin receptor regulate translation of the respective mRNAs. These
elements either destabilize the mRNA (transferrin receptor) or allow translation of
the mRNA (ferritin) when iron levels are high.