The Cap is recognized in the cytoplasm by special initiation factors to allow assembly of a ribosome at the mRNA initiation site. Because the initiation of translation involves Cap recognition, each protein in eukaryotic cells is encoded on its own mRNA. The capped base of an mRNA corresponds to the start site of transcription of the precursor RNA from which it is processed.

From the article on genes.

A retrotransposon (another kind of interspersed repeat) is transcribed normally, then reverse-transcribes to form dsDNA that integrates the same way that a tansposon does: a staggered nick; insertion; filling in of the 5′ and 3′ ssDNA. The upstream LTR acts as a promoter and the downstream LTR contains a poly-A site to produce transcripts from the integrated element. Between them is a coding region that encodes proteins for transposition and also acts as a copy template.

Lastly, a non-LTR retrotansposon is the same as an LTR retransposon but has a different method for integration.

With non-viral retrotransposons, if a LINE has a weak poly(A) signal, then sometimes transcription will continue and include an adjacent 3? gene (eventually terminating at that gene’s strong poly(A) signal). ORF2 then reverse-transcribes the RNA transcript of the LINE and gene, eventually inserting the gene at a new location along with the SINE in a phenomenon known as exon shuffling.

Exon shuffling can occur double crossover between interspersed repeats.

Also, via DNA transposons.

A non-LTR retrotransposon, conversely, integrates as follows:

DNAse sensitivity is an effective way to determine if a gene is condensed or decondensed form. A condensed gene will be more resistant to DNAse than a gene that is not condensed. Afterward, decondensation of the entire genome and PCR analysis will reveal what has and has not been digested.

The cDNA microarray experiment identified gene regulation at the transcriptional level. cDNA is single-stranded DNA that has been reverse-transcribed in vitro from cellular mRNA. By soaking a cDNA microarray with labeled RNAs, it is possible to determine which genes are being transcribed in a particular cell; only active genes were being transcribed. Thus, regulation is at the transcriptional level.

The Cleavage/Polyadenylation reaction is carried out the polyadenylation complex, a large multi-protein complex assembled from CPSF, CStF, CF1, and CF2. Some pre-mRNA’s have multiple poly(a) signals, and cleavage and polyadenylation at these different signals can include or exclude various 3′ terminal exons. Ser2 on the CTD is phosphorylated

TFIIH is a large General Transcription Factor (GTF) of 9 subunits, nearly as large as Pol II. Two of the subunits are homologous to DNA helicases, enzymes that use energy from ATP hydrolysis to separate the strands of a DNA double helix. One of the subunits is a kinase that phosphorylates Ser5 of the Pol II large subunit C-terminal domain (CTD) heptapeptide repeat (Tyr-Ser-Pro-Thr-Ser-Pro-Ser).

Coupling 3′ cleavage & transcription termination:

B lymphocytes carry IgM, and upon exposure to their complementary antigen become antibody-secreting plasma cells. This switch is accomplished by a change in the IgM mRNA poly(A) site. There are two poly(A) sites, with the upstream site requiring more CStF. In B cells, CstF64 levels are low; in plasma cells, CStF64 expression is unregulated.

Exon Definition (aka Exon Recognitino) is performed by the Cross Exon Recognition Complex (CERC).

Two general molecular mechanisms are to: repress exon inclusion (as Sxl does); and promote exon inclusion (as Tra does). Sxl sits on L3 and blocks assembly of spliceosome components, thus causing the entire exon to get skipped. Tra stabilizes the exon recognition complex by binding SR proteins, thus causing an exon to get included that otherwise would not have been.

Sxl sits on L3, forming a functional L1-L2-L4 Sxl protein. This Sxl sits on Tra’s 2^nd exon and a function Tra protein is expressed composed of Exons 1 and 3. Rbp1 and Tra2 sit at the 5′ end of the 4^th Dsx exon, promoting splicing at that region and thus inclusion of Exon 4 (to encode a functional Dsx protein).

Transport is unidirectional because of rapid dissociation of importin. Ran-GTP is only hydrolysed to Ran-GDP in the cytoplasm, favoring release of Ran and binding of cargo.

Nuclear transport factor 2 (NTF2) binds Ran-GDP and FG-nucleoporins to return Ran-GDP back to the nucleus.

Translation can be controlled by cytoplasmic polyadenylation. Certain mRNAs are made to have short poly(A) tails and thus no translation initiation. These are localized at synapses, far away from that cell’s nucleus. Synaptic activity will stimulate polyadenylation in the immediate region of the synapse.

This mechanism has been determined for the egg, and is likely similar for neurons:

RNA interference pathways (RNAi) are mediated by small sequence-specific RNA molecules that shut down gene expression by one of three mechanisms: translational repression; mRNA degradation; and heterochromatin formation. Cleavage is a sort of rapid degradation by both 5′-3′ and 3′-5′ exonucleases. A targeted mRNA has two possible outcomes:

Dicer, which cleaves long dsRNA into short fragments of ~20 nucleotides. One of the two strands of each fragment, known as the guide strand, is then incorporated into the RNA-induced silencing complex (RISC) (promoted by Dicer).

A pre-miRNA consists of the RNA, Drosha and Pasha. Primary miRNA transcript cropped in nucleus by enzyme Drosha. Exported by exportin 5. dsRNA portion of this pre-miRNA is bound and cleaved by Dicer to produce the mature miRNA molecule that can be integrated into the RISC complex

Thus, miRNA and siRNA share the same cellular machinery downstream of their initial processing.

If translation terminates >50 nt upstream of an EJC, it is not displaced and signals for that mRNA to be degraded. Nonsense mutations generally lead to an early stop codon, so this means that most exonic nonsense mutation will be degraded. The EJC recruits a protein called UPF1 to the mRNA. This in turn stimulates binding of decapping enzymes and deadenylation, leading to rapid degradation. Nonsense mediated decay is advantageous because it prevents the production of truncated and potentially dominant negative mutant proteins in the heterozygous state.

Note that the normal termination codon is almost always in the last exon of a gene. Nonsense mutations in this exon do not lead to nonsense mediated mRNA decay and produce slightly truncated proteins.

CDKs are controlled at every level: presence (controlled degradation); activity (inhibitors); and even specificity (cyclins). Examples are shown below:

SCF and APC/C are ubiquitin protein ligase complexes that that control three major transitions in the cell cycle: onset of S-phase through degradation of Sic1 by SCF; initiation of anaphase via degradation of securin by APC-Cdc20; and exit from mitosis via degradation of mitotic cyclins by APC-Cdh1. APC has several substrates that must be degraded at different times in the cycle; thus, its activity is directed by specificity factors that bind it. SCF only degrades Sic1 and thus its activity is regulated only by phosphorylation of its substrate.

In the spindle assembly checkpoint (aka metaphase checkpoint), mitotic arrest deficient 2 (aka Mad2) blocks metaphase until every single kinetochore has properly attached to spindle microtubules. Mad2 exists in an open conformation (Mad2^O) and a closed conformation (Mad2^C).

1. Mad1 and Mad2^C form a tetramer that binds unattached kinetochores via the Mad1 subunit.
2. Mad2^C of the kinetochore-bound tetramer can transiently bind Free Mad2^O.
3. This transient interaction causes Mad2^O to bind and inactivate Cdc20, and to convert from Mad2^O→Mad2^C.
4. Mad2^C-Cdc20 transiently interacts with additional free Mad2^O.
5. This causes Mad2^O to bind and inactivate additional Cdc20, forming a second Mad2^C-Cdc20.
6. The cycle repeats and free Mad2^O is quickly converted to Mad2^C-Cdc20.
7. Binding of microtubules to the kinetochore displaces Mad1-Mad2^C tetramers.
8. Free tetrameric Mad2^C cannot bind Mad2^O. Instead, free tetrameric Mad2^C binds p31^comet.
9. p31 then binds the Mad2^C of the Mad2^C-Cdc20 complexes, resulting in release of active Cdc20.

Just a few Mad1-Mad2^C tetramers bound to kinetochores can generate enough Mad2^C-Cdc20 to overcome p31 activity. Once all kinetochores have attached to microtubules (thus releasing all Mad1-Mad2^C tetramers), p31 activity predominates and active Cdc20 is released from Mad2^C. The active Cdc20 binds APC/C, and the APC/C-Cdc20 degrades securin, the inhibitor of separase. Free separase digests the Kleisin subunit of cohesin, breaking open the Smc1-Smc3-Kleisin ring and allowing sister chromatids to separate. Cohesin is a Smc1/Smc3/Kleisin heterotrimer that holds together sister chromatids, with Kleisin acting like a clasp.

As anaphase ends, dephosphorylation of existing lamin begins. Once the genetic material has fully segregated at the completion of anaphase, production of new lamin is well underway. The new lamin drags tubes of smooth endoplasmic reticulum across the surface of the chromatin; these tubes flatten and merge, forming a solid nuclear membrane. The endoplasmic reticulum and lamin detach themselves from the chromatin. In the mature daughter cell, the lamina is a continous layer that is bound to the inner membrane of the nuclear envelope by emerin proteins.

G1 cyclin-CDK inactivates human Cdh1 and human inhibitors of cyclin-dependent kinase 4 and 6 (INK4s), important tumor suppressors that inhibit passage through G1 by inhibiting the mid G1 cyclin-CDKs. Both genes encoding INK4a are mutated in many human tumors, as they are less able to inhibit passage into G1.